JP5146835B2 - Galectin composition and method of use thereof in the treatment of dry eye syndrome - Google Patents

Description

  Provided are galectin protein compositions, methods and kits for the treatment of dry eye syndrome.

  Dry eye syndrome (DES) is a common symptom, with as many as 10% of the 30- to 45-year-old population showing symptoms, increasing over the age of 45, and 20% of the population showing symptoms. (Schein et al., 1997, Am J Ophthamol 124, 723-72; Brewitt and Sistani, 2001, Surv of Ophthalmol 45: S119-S202). DES causes eye irritation, blurred vision and visual field vibration and increases the risk of visual acuity for corneal infections and ulcers. The histological effects of DES include aberrant proliferation and differentiation, with a decrease in conjunctival goblet cell density and a decrease in mucus production and abnormalities in the ocular surface epithelium (Murillo-Lopez and Pflugfelder, 1996, Dry Eye. In: Krachmer J, Mannis M, Holland E, eds. The Cornea. Mosby, St. Louis, MO. 663-686; Dursun et al., 2002, Invest Ophthalmol & Vis Sci 43: 632-638 ). Dry eye disease is a chronic disease. Symptoms and signs are greatly influenced by environmental factors such as humidity and air movement, but it is also greatly influenced by the degree of viewing work such as reading and using computers.

Typical symptoms of DES include burning pain, itching, foreign body sensation, stinging, dryness, photophobia, eye fatigue, and redness. Dry eye disease is a chronic disease. Symptoms and signs are greatly influenced by environmental factors, such as humidity and air movement, but are also greatly influenced by the degree of viewing tasks such as reading and using computers (Rheinstrom, 1999 In Yanoff, ed. Ophthalmology. 1 ^st Ed. Editor. Mosley International Ltd, St Louis, MO; Foulks, 2003, The Ocular Surface, 1: 20-30).

  A wide variety of artificial tear products are on the market, all of which only temporarily relieve symptoms. At present, there are no treatments that improve symptoms. Accordingly, there is a need in the art for additional pharmaceuticals and compositions that treat dry eye syndrome. In particular, there is a need for drugs, compositions and treatment methods.

Schein et al., 1997, Am J Ophthamol 124, 723-72 Brewitt and Sistani, 2001, Surv of Ophthalmol 45: S119‐S202 Murillo‐Lopez and Pflugfelder, 1996, Dry Eye. In: Krachmer J, Mannis M, Holland E, eds. The Cornea. Mosby, St. Lois, MO. 663-686 Dursun et al., 2002, Invest Ophthalmol & Vis Sci 43: 632-638 Rheinstrom, 1999, Dry eye.In Yanoff, ed.Ophthalmology.1st Ed.Editor.Mosley International Ltd, St Louis, MO Foulks, 2003, The Ocular Surface, 1: 20-30

  The present invention is a method of treating dry eye in a mammal in need of treatment, characterized in that a therapeutically effective amount of galectin protein is administered to the mammal. In one embodiment, a method for preventing eye dryness in a mammal is mentioned, which comprises identifying a mammal in need of dry eye prevention and administering a therapeutically effective galectin protein to the identified mammal. Therefore, the method is used for mammals in need of treatment or prevention, at least mammals that are ocular epithelial wounds, mammals that are before use of antihistamines, mammals that are before use of anti-inflammatory agents, excimer laser treatment It is used for the treatment or prevention of mammals that apply to any of the mammals before administration. For example, the mammal includes a human. Furthermore, the galectin protein is selected from galectin-8, galectin-7 or galectin-3. In certain embodiments, dry eye is a persistent syndrome. For example, dry eyes cause epithelial erosion, which further leads to corneal wounds.

  Generally, the galectin-8 protein used in the above method comprises the amino acid sequence of SEQ ID NO: 4 or 5. For example, the galectin-8 protein comprises an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO: 4 or 5. As used herein, “substantially identical” means that galectin-8 is at least 60% identical, 70% identical, 80% identical to the amino acid sequence of SEQ ID NO: 4 or 5. Or 90% identity.

  In general, the galectin-7 protein used in the method described above comprises the amino acid sequence of SEQ ID NO: 2. For example, galectin-7 protein comprises an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO: 2. Galectin-7 has at least 60% identity, 70% identity, 80% identity, or 90% identity with the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the galectin-7 protein comprises a galectin-7 galactosidic binding region.

  Generally, the galectin-3 protein used in the above method comprises the amino acid sequence of SEQ ID NO: 1. For example, the galectin-3 protein comprises an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO: 1. Galectin-3 has at least 60% identity, 70% identity, 80% identity, or 90% identity with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the galectin-3 protein comprises a galectin-3 galactosidic binding region.

  Another embodiment of the present invention includes a pharmaceutical composition having an effect of promoting the treatment of dry eye, the pharmaceutical composition comprising a pharmaceutically suitable carrier or diluent, and conjunctival and / or corneal epithelium. Provide a sufficient amount of galectin-8 protein to promote health. Other embodiments of the present invention include pharmaceutical compositions that have an accelerating effect on the treatment of dry eye, the pharmaceutical composition comprising a pharmaceutically suitable carrier or diluent, and conjunctival and / or corneal epithelium. A sufficient amount of galectin-7 protein to promote the health of Other embodiments of the present invention include pharmaceutical compositions that have an accelerating effect on the treatment of dry eye, the pharmaceutical composition comprising a pharmaceutically suitable carrier or diluent, and conjunctival and / or corneal epithelium. A sufficient amount of galectin-3 protein to promote the health of Other embodiments of the present invention include pharmaceutical compositions that have an accelerating effect on the treatment of dry eye, the pharmaceutical composition comprising a pharmaceutically suitable carrier or diluent, and conjunctival and / or corneal epithelium. A sufficient amount of galectin-8 protein to promote the health of In any of these pharmaceutical compositions, dry eye is dry eye disease with recurrent epithelial erosion. For example, dry eye causes a wound, which is a corneal wound. In addition, dry eyes are caused by excimer laser keratotomy.

  In various pharmaceutical composition examples, the galectin-8 protein comprises the amino acid sequence of SEQ ID NO: 4 or 5. Alternatively, the galectin 8 protein comprises an amino acid sequence that is substantially identical to the amino acid sequence of amino acid number 4 or 5. Alternatively, the galectin-8 protein includes a galectin-8 N-terminal region and a region rich in galectin-8 proline, glycine, and tyrosine. Alternatively, the galectin-8 protein comprises a region rich in galectin-8 proline, glycine and tyrosine and a galectin-8 galactoside binding region. For example, galectin-8 protein contains a galectin-8 galactoside binding region. Alternatively, the galectin-7 protein comprises the amino acid sequence of SEQ ID NO: 2. For example, galectin-7 protein comprises an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO: 2. Alternatively, the galectin-7 protein includes a galectin-7 galactoside binding region. Alternatively, the galectin-3 protein comprises the amino acid sequence of SEQ ID NO: 1. For example, galectin-3 protein comprises an amino acid sequence that is substantially identical to the amino acid sequence of amino acid number 1. For example, a galectin-3 protein includes a galectin-3 galactoside binding region.

  The present invention is further characterized by a method of treating dry eye in a mammal in need of treatment, comprising the step of administering a therapeutically effective amount of the substance on the corneal or conjunctival surface. Affects tear film diffusion. Further, the present invention is a method for treating dry eye disease in a mammal in need of treatment, comprising the step of administering a therapeutically effective amount of a substance in a mammal suffering from dry eye disease, the substance comprising galectin -8 affects the expression of protein. For example, the substance comprises a galectin-8 protein having the amino acid sequence of SEQ ID NO: 4 or 5. For example, the substance comprises a galectin-8 protein having an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 4 or 5.

  The present invention refers to various patents, scientific articles, and other publications. The contents of each item are incorporated herein by reference. The contents of all websites referred to here (as of the present application) are also included in this specification by referring to them.

  The examples of the present invention are based on the concept that the carbohydrate binding protein galectin promotes tear film diffusion in the cornea and conjunctival epidermis. Galectins are galactose binding proteins that bind with high affinity to the Galβ1-4GlcNAC disaccharide found in mucin O-linked oligosaccharides. Most galectins are bivalent or multivalent, and galectins promote the diffusion of the tear film by binding oligosaccharide chains of secretory mucins and transmembrane mucins (other glycoproteins). The transmembrane mucin (other glycoprotein) is present on the outermost surface of the cornea and conjunctival epithelium.

  The present invention provides a pharmaceutical composition comprising galectin-8, galectin-3 and / or galectin-7 and is useful in promoting reepithelialization of wounds in damaged mammalian tissue. The present invention further provides a method of therapeutic treatment in epithelial damaged mammalian tissue, wherein the method provides a therapeutically effective amount of galectin-1, galectin-3, galectin-7, galectin to a mammal suffering from epithelial damage. -8, or a mixture of at least two of Galectins-1, -3, -7, -8. When a mixture of galectin-1, -3, -7, -8 is administered, any of the aforementioned galectins can be administered before, in combination with or after administration of the other galectins.

  The present invention encompasses the discovery that galectin-3 is up-regulated in corneal epithelial cells to the cornea with injury (Example 1). The present invention also includes the discovery that re-epithelialization of corneal transepithelial excimer laser wounds and corneal alkali burns is significantly slower in galectin-3-deficient mice than in wild type mice (Example 2). . The present invention further provides the discovery that in galectin-3-deficient mice, the expression of many damage-related genes (eg, toroid-like protein and galectin-7) is abnormal (Example 3). Furthermore, the present invention also shows that exogenous galectin-3 and galectin-7 promote re-epithelialization of corneal wounds (Examples 4, 5 respectively).

<Galectin>
A lectin is a protein, and is clarified in particular by its ability to bind to carbohydrates and to aggregate cells (see, for example, Sharon, Trends Biochem. Sci. 18: 221, 1993). Lectins have been shown to be responsible for a wide variety of cell functions, including cell-cell and cell-matrix interactions. Lectins are found in a wide range of plants, invertebrates and mammals. Animal lectins are divided into four different groups, including 1) C-type lectins, 2) P-type lectins, 3) Galectins (formerly called S-type lectins), and 4) Pentraxins. (For example, Barondes et al., J. Biol. Chem. 269: 20807, 1994).

  All mammalian galectins have been analyzed in detail for recognition of β-lactose and β-galactoside related factors. All mammalian galectins have similar affinities for small β-galactosides, whereas for more complex complex polysaccharides there is a marked difference in binding specificity (Henrick et al., Glycobiology 8; 45 Sato et al., J. Biol. Chem. 267: 6983, 1992; Seetharaman et al., J. Bioil. Chem. 273: 13047, 1998). In addition to binding to β-galactoside sugars, galectins have hemagglutination activity. Laminin is a glycoprotein that exists in nature, has many polylactosamine chains, and has been shown to be a natural ligand for specific galectins. Laminin is a component of the basal layer and extracellular matrix, which is below all epithelium and surrounds individual muscle, fat, and Schwann cells. Cell-basal layer interactions are known to affect the transport and / or differentiation of various cell types during mammalian growth. Galectins do not have a native sequence that specifies membrane movement, but are secreted and present in the cell. In addition to affinity for β-galactoside sugars, galectin family members share significant sequence similarity in the carbohydrate recognition region (also referred to as CRD, or protein binding region). The amino acid residues associated with the carbohydrate recognition region have been determined by X-ray crystallography (Lobsanov et al, J. Biol. Chem. 267: 27034, 1993 and Seethnaraman et al., Supra). Galectins are believed to be involved in a wide variety of biological functions, including cell adhesion (Copper et al., J. Cell Biol. 115: 1437, 1991), growth regulation (Wells et al., Cell 64:91 1991), cell migration (Hughes, Curr. Opin. Struct. Biol. 2: 687, 1992), malignant transformation (Raz et al., Int. J. Cancer 46: 871, 1990) and immune response (Offner et al. al, J. Neuroimmunol. 28: 177, 1990). Currently, 12 eukaryotic members of the galectin family have been characterized.

<Galectin-3>
Galectin-3 family member proteins (formerly known as CBP-35, Mac-2, L-34, εBP and RL-29) typically contain about 240 to 270 amino acids and have a molecular weight of It is in the range of about 25 to 29 kDa. Galectin-3 proteins generally consist of a short N-terminal region, a C-terminal region containing a galactoside binding region, and a region rich in intervening proline, glycine and tyrosine. The intervening proline, glycine and tyrosine-rich region contains 7 to 10 conserved amino acid sequence repeats (Liu et al., Biochemistry 35: 6073, 1996 and Cherayil et al., Proc. Natl. Acad. Sci. USA, 87: 7324, 1990). The tandem repeats are similar to those found in the collagen gene superfamily. The number of repeats varies from galectin-3 protein, accounting for differences in galectin-3 protein size by different species. The galectin-3 N-terminal region binds to the surface containing the glycoconjugate ligand and multimerizes the protein.

  Galectin-3 is expressed in various inflammatory cells (active macrophages, basophils and mast cells) and in epithelia and fibroblasts of various tissues (Perillo et al., J. Mol. Med. 76: 402 , 1998). Galectin-3 is found on the cell surface, extracellular matrix (ECM), cytoplasm and cell nucleus. Galectin-3 is said to mediate cell-cell and cell-matrix interactions on the cell surface or at the ECM, which are made by binding complementary glycoconjugates. The glycoconjugate contains polylactosamine chains that are often found in ECM and cell surface molecules. Galectin-3 is said to inhibit cell-matrix adhesion by binding to lamin. In the cell nucleus, galectin-3 is a known cell adhesion molecule (eg, α6β1 and α4β7 integrins, Warlfield et al., Invasion Metastasis 17: 101, 1997 and Matarrese et al., Int. J. Cancer 85: 545, 2000). And by controlling the expression of cytokines (eg, IL-1, Jeng et al., Immunol. Lett. 42: 113, 1994), they can also participate indirectly in cell-matrix interactions. In certain organs such as the kidney, galectin-3 expression is developmentally regulated, and the expression level of galectin-3 in alveolar epithelial cells and hepatocytes is up-regulated with injury. In the growth process, galectin-3 has been shown to condense in the nuclei of certain cell types. Galectin-3 expression is increased in certain tumors, suggesting that galectin-3 is involved in metastasis. Indeed, overexpression of galectin-3 in weakly metastatic cell lines significantly increases metastatic potential (Raz et al., Supra).

  Human galectin-3 is 250 amino acids long and has a molecular weight of approximately 26.1 kDa (SEQ ID NO: 1, FIG. 1). As shown in FIGS. 1, 3, 5 and 7, human galectin-3 has the following regions, characteristic sequences or other structural features (for general information on PS and PF prefix recognition numbers: Sonnhammer et al., Protein 28: 405, 1997). N-terminal region around amino acid residues 1 to 14 of SEQ ID NO: 1, region rich in proline, glycine and tyrosine around amino acid residues 15 to 116 of SEQ ID NO: 1, amino acid residue 117 of SEQ ID NO: 1 A galactoside binding region around 247 to 247, a galaptin characteristic sequence around amino acid residues 181 to 200 of SEQ ID NO: 1 (PROSITE number, PS00309), one around amino acid residues 4 to 7 around SEQ ID NO: 1 Two potential N-glycosylation sites (PROSITE number, PS00001), two potential protein kinase C phosphorylation sites (PROSITE number, around amino acid residues 137 to 179 and 194 to 196 of SEQ ID NO: 1, PS00005), amino acid residues 6 to 9 and 175 to 178 of SEQ ID NO: 1 Two potential casein kinase II phosphorylation sites (PROSITE number, PS00006) and amino acid residues 24 to 29, 27 to 32, 34 to 39, 43 to 48, 52 to 57, 61 of SEQ ID NO: 1. 8 potential myristoylation sites (PROSITE number, PS00008) around 66, 65-70 and 68-73.

  As defined herein, a galectin-3 protein comprises a “galectin-3 N-terminal region”, a galectin-3 “region rich in proline, glycine and tyrosine” and / or a galectin-3 “galactoside binding region”. . These areas are further defined as follows.

  As used herein, galactin-3 “N-terminal region” includes an amino acid sequence of about 10 to 20 amino acids, preferably about 14 amino acids, of 1 to 14 of SEQ ID NO: 1. It shall have at least 60%, 70%, 80%, 90%, 95%, 99% or 100% identity with the amino acid. The N-terminal region can include an N-glycan formation site (PROSITE number, PS00001) and / or a casein kinase II phosphorylation site (PROSITE number, PS00006). The N-sugar chain forming site of PROSITE has N- {P}-[ST]-{P} as a common sequence, and the casein kinase II phosphorylation site has [ST] -X (2) as a common sequence. -Have [DE]. In the above consensus sequences and other motifs or characteristic sequences described herein, the single letter symbols for amino acids that are IUPAC standards are used. Each element of the format is separated by a dash (-), square brackets ([]) indicate the specific residues allowed at each position, and X indicates any residues allowed at each position. The numbers in parentheses (()) indicate the number of residues indicated by the accompanying amino acid. In some embodiments, the N-terminal region includes amino acids L7 and L11 of SEQ ID NO: 1. As shown in FIG. 3, these amino acids are conserved across various mammalian species of galectin-3, which amino acids play a catalytic and / or structural role.

  As described herein, a “proline, glycine and tyrosine rich region” of galectin-3 comprises an amino acid sequence of about 60 to 140 amino acids, more preferably about 80 to 120 amino acids, or about 90 From about 110 to 110 amino acids and has about 70%, 80%, 90%, 95%, 99% or 100% identity with 15 to 116 amino acids of SEQ ID NO: 1. The region rich in proline, glycine and tyrosine can also contain 1,2,3,4,5,6,7 or 8 N-myristoylation sites (PROSITE number, PS00008), said N-myristoyl The conjugation site has G- {EDRKHPFYW} -X (2)-[STAGCN]-{P} as a common sequence. In one embodiment, the region rich in proline, glycine and tyrosine comprises the following amino acids and regions of SEQ ID NO: 1. The sequences and regions include G21, P23, G27, N28, P30, G32, G34, P37, Y41-P46, G53, Y55-G57, P61, G62, G66, P72, G73, G77, Y79-G81, P83, G87, Y89, P90, G99, Y101, P102, P106, Y107, A109, L114, and V116. These amino acids and regions are conserved across various mammalian species of galectin-3, which amino acids and regions play a catalytic and / or structural role. (Refer to the amino acids indicated by “*” in FIG.

  As described herein, the “galactoside binding region” of galectin-3 comprises an amino acid sequence of about 80 to 180 amino acids, which is a sequence with the consensus sequence PF00337 registered in PFAM (No. 3). In comparison, it has a bit score of at least 150. Preferably, the galactoside binding region of galectin-3 comprises at least about 100 to 160 amino acids, more preferably comprises 110 to 150 amino acids or about 120 to 140 amino acids and is registered with PFAM (No. 3). In comparison with the common sequence PF00337, the bit score is at least 150, more preferably at least 175, and even more preferably 200 or more.

In order to calculate the bit score in the sequence comparison between a specific sequence and the common sequence PF00337 registered in PFAM (No. 3), the target sequence is calculated using the MMAM PFAM database (eg, PFAM database, release 2.1). ) And the numerical value obtained at www.sanger.ac.uk/Sogtware/Pfam . Is used as the initial variable. A description of the PFAM database is described in Sonnhammer et al., Supra, and details of HMMs can be found, for example, in the literature (Griboskov et al., Meth. Enzymol. 183: 146, 1990 and Stulz et al., Protein Sci 2: 305, 1993).

  The galactoside binding region of galectin-3 is further divided into one, preferably two protein kinase C phosphorylation sites (PROSITE number, PS00005), casein kinase II phosphorylation site (PROSITE number, PS00006) and / or galactin characteristic sequences (PROSITE). Number, PS00309). The protein kinase C phosphorylation site has the following consensus sequence: [ST] -X- [RK]. The characteristic sequence of galaptin is the following consensus sequence: W- [GEK] -X- [EQ] -X- [KRE] -X (3,6)-[PCTF]-[LIVMF]-[NQEGSKV] -X- [ GH] -X (3)-[DENKHS]-[LIVMFC]. In one implementation, the galactoside binding region of galectin-3 is the next amino acid and region of SEQ ID NO: 1, P117, Y118, L120-L122, G125, P128, R129, L131-I134, G136-V138, N141, N143, R144, L147, F149, R151, G152, D154, A156-F163, E165, R169-N174, N179-G182, E184-R186, F190-E193, G195, P197-K199, Q201-L203, E205, D207-Q220, N222, R224, L228, I231, I236, G238-I240 and L242-S244. These amino acids and regions are conserved across various mammalian species of galectin-3, which amino acids and regions play a catalytic and / or structural role. (See amino acids marked with “*” in Figure 3)

  One galectin-3 protein of the present invention comprises the amino acid sequence of human galectin-3 shown in SEQ ID NO: 1. Another galectin-3 protein of the present invention has an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 1. “Substantially identical” as used herein refers to a first amino acid comprising a sufficient or minimal number of amino acid residues, which amino acid residues are the residues of the aligned amino acid sequence of the second amino acid sequence. It is assumed that the first and second amino acid sequences can have a common structural region and / or a common functional activity. For example, at least 60% or 65% identity to SEQ ID NO: 1, preferably at least 75% identity, more preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98% or 99% of the amino acid sequence comprising a common structural region with identity is substantially identical to the amino acid sequence of SEQ ID NO: 1. In particular, proteins containing accidental or intentionally induced mutations such as deletions, additions, substitutions or variations in specific amino acid residues of SEQ ID NO: 1 are included in the definition of galectin-3 as described herein. . It will also be appreciated that the galectin-3 protein may include a region represented by the amino acid sequence of galectin-3 of other mammalian species, as defined herein. Other mammalian species include, but are not limited to, cows, dogs, cats, goats, sheep, pigs, mice and horses.

  Calculation of sequence identity between sequences is performed as follows. To determine the percent identity of two amino acid sequences, the sequences are first aligned in order to make an optimal comparison (eg, one amino acid sequence or both the first and second amino acid sequences in an optimal comparison). Difference is incorporated). Then, the amino acid residues at corresponding amino acid sites or nucleotide sites are compared. If the site of the first sequence is the same amino acid residue or nucleotide as the corresponding site of the second sequence, the protein is identical at that site. The percent identity between two sequences is determined by the number of sites that are identical between the sequences and takes into account the number of differences and the length of each difference. The number of differences and the length of each difference are required for optimal sequence comparison of the two sequences.

  Sequence comparison between two sequences and determination of percent identity is done using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined by a sequence comparison software program using initial value variables. Suitable programs include, for example, CLUSTAL W (Thompson et al., Nuc. Acids Research 22: 4673, 1994 (www.ebi.ac.uk/clustalw)), BL2SEQ (Tatusova and Madden, FEMS Microbiol. Lett. 174 : 247, 1999 (www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html)), SAGA (Notredame and Higgins, Nuc. Acids Research 24: 1515, 1996 (igs-server.cnrs-mrs.fr / ~ cnotred)) and DIALIGN (Morgenstern et al., Bioinformatics 14: 290, 1998 (bibiserv.techfak.uni-bielefeld.de/dialign)).

(www.ncbi.
<Galectin-7>
Galectin-7 family member proteins usually exist as monomers, contain 130 to 140 amino acids, and have a molecular weight in the range of about 15 to 16 kDa (eg, Magnaldo et al., Develop. Biol. 168: 259, 1995 and Madsen et al., J. Biol. Chem. 270: 5823, 1995). Galectin-7 expression is associated with the initiation of epithelial layer formation (Timmons et al., Int. J. Dev. Biol. 43: 229, 1999). Galectin-7 is thought to be responsible for cell-matrix and cell-cell interactions. Galectin-7 is found in areas where cells are in contact with each other (eg, the upper layer of the human epidermis), and the expression of galectin-7 is highly down-regulated in anchorage-independent keratinocytes, and malignant keratin Not seen in producer cell lines. Galectin-7 is required for the maintenance of normal keratinocytes (see Madsen et al., Supra).

  Human galectin-7 contains 136 amino acids and has a molecular weight of about 15.1 kDa (SEQ ID NO: 2, FIG. 2). As shown in FIGS. 2, 4, 6 and 8, human galectin-7 includes the following regions, characteristic sequences, or other structural features. A galactoside binding region located around amino acid residues 5 to 135 of SEQ ID NO: 2, a galaptin characteristic sequence located around amino acid residues 70 to 89 of SEQ ID NO: 2 (PROSITE number, PS00309), amino acids of SEQ ID NO: 2 One N-glycosylation site located around residues 29 to 32 (PROSITE number, PS00001), one protein kinase C phosphorylation site (PROSITE) located around amino acid residues 132 to 134 of SEQ ID NO: 2. No. PS00005), one casein kinase II phosphorylation site (PROSITE number, PS00006) located around amino acid residues 9 to 12 of SEQ ID NO: 2, and amino acid residues 13 to 18 and 44 to 49 of SEQ ID NO: 2. Two myristoylation sites (PROS) located around TE number, is PS00008).

  As defined herein, a “galectin-7 protein” comprising galectin-7 comprises a galactoside binding region. This area is further defined as follows.

  As used herein, the galactoside binding region of galectin-7 consists of an amino acid sequence of about 80 to 180 amino acids and is at least 80 by sequence comparison with the consensus sequence PF00337 of PFAM (SEQ ID NO: 3). With a bit score of. Preferably, the galactoside binding region of galectin-7 consists of at least about 100 to 160 amino acids, more preferably about 110 to 150 amino acids, or about 120 to 140 amino acids, and comprises PFAM (SEQ ID NO: 3). The sequence comparison with the common sequence, PF00337, has a bit score of at least 80, more preferably at least 100, and even more preferably 120 or more. The galactoside binding region of galectin-7 consists of one N-glycan formation site (PROSITE number PS00001), one protein kinase C phosphorylation site (PROSITE number PS00005), one casein kinase II phosphorylation site (PROSITE number, PS00006). ) One or two myristoylation sites (PROSITE number, PS00008) and / or galeptin characteristic sequences (PROSITE number, PS00309). In some embodiments, the galactoside binding region of galectin-7 comprises the following amino acid sequence or region of SEQ ID NO: 2. M1, S2, H6, K7, L10, P11, G13, R15, G17-V19, R21-G24, V26, P27, A30, R32-Q43, D46-N63, K65, Q67, G68, W70-G76, G78, P80-L90, I92, G97-K99, V101, G103, D104, Y107, H109, F110, H112, R113, P115, V119, R120, V122-L130, S132, I135, and F136. These amino acids and regions are conserved in some mammalian galectins-7 and play a catalytic and / or structural role (see amino acids labeled with “*” in FIG. 4).

  One galectin-7 protein of the present invention comprises the human galectin-7 amino acid sequence set forth in SEQ ID NO: 2. Another galectin-7 protein of the present invention comprises an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 2. In particular, proteins that include accidental or intentionally induced mutations, such as deletions, additions, substitutions, or modifications at specific amino acid residues of SEQ ID NO: 2, fall within the definition of galectin-7 of the present invention. It will also be appreciated that the galectin-3 protein may include a region represented by the amino acid sequence of galectin-3 of other mammalian species, as defined herein. Other mammalian species include, but are not limited to, cows, dogs, cats, goats, sheep, pigs, mice and horses.

<Galectin-8>
Galectin-8 is a widely expressed protein, eg, found in the liver, heart, muscle, kidney, spleen, hind limbs and brain, and human and rat galectin-8 gene and protein sequences are available ( For example, see Hadari, et al., Trends in Glycosci and Glycotechnol. 9: 103-112, 1997). Due to its high hydrophilicity and ability to bind to Galβ1-4GlcNAC disaccharide found in mucin O-linked oligosaccharides, the protein is an ideal therapeutic agent for eye syndrome.

  There are two known amino acid sequences of human galectin-8, the 316 amino acid type (accession number O00214, created on November 1, 1997) and the 359 amino acid type (accession number Q8TEV1, June 1, 2002). Creation). While these sequences are similar or identical in considerable length, there are some different parts overall. The 316 type amino acid sequence is shown below using the single letter amino acid symbol (SEQ ID NO: 4).

The long amino acid sequence is shown below (SEQ ID NO: 5).

  As defined herein, “galectin-8 protein” refers to “N-terminal region” of galectin-8, “region rich in proline, glycerin and tyrosine” of galectin-8, and / or galectin-8. Of the “galactoside binding region”.

  As described herein, the “N-terminal region” of galectin-8 comprises an amino acid sequence of about 10 to 20 amino acids, preferably about 14 amino acids, 60%, 70% with the amino acids of SEQ ID NO: 5 or 80%, 90%, 95%, 99%, or 100% identity. The N-terminal region includes an N-glycan formation site (PROSITE number, PS00001) and / or a casein kinase II phosphorylation site (PROSITE number, PS00006). The N-glycosylation site of PROSITE has N- {P}-[ST]-{P} as a common sequence, and the casein kinase II phosphorylation site by PROSITE is [ST] -X (2)-[ DE]. The common sequence includes other motifs or characteristic sequences.

  As used herein, a “proline, glycine and tyrosine rich region” of galectin-8 comprises an amino acid sequence of about 60 to 140 amino acids, more preferably 80 to 120 amino acids, or 90 to 110. With 60%, 70%, 80%, 90%, 95%, 99%, or 100% identity with 15 to 116 amino acids of each amino acid of SEQ ID NOs: 4 and 5. Regions rich in proline, glycine and tyrosine can also have 1, 2, 3, 4, 5, 6, 7 or 8 N-myristoylation sites (PROSITE number, PS0008), and the myristoylation sites are common It has G- {EDRKHPFYW} -X (2)-[STAGCN]-{P} as an array. In one embodiment, the region rich in proline, glycine and tyrosine comprises the following amino acids and regions of SEQ ID NO: 4. Amino acids and regions are G20, P23, P28, G29, G36, P39 and other residues apparent to those skilled in the art. These amino acids and regions are conserved in several mammalian galectins-8 and play a catalytic and structural role. In one example, the proline, glycine and tyrosine rich region comprises the following amino acids and regions of SEQ ID NO: 5. Amino acids and regions are G21, P24, P29, G30, G37, P40 and other residues apparent to those skilled in the art.

  As used herein, the “galactoside binding region” of galectin-4 consists of an amino acid sequence of about 80 to 180 amino acids and has a bit score of at least 150 when compared with PF00337, a consensus sequence of PFAM (SEQ ID NO: 3). have. Preferably, the galectin-3 galactoside binding region comprises at least about 100 to 160 amino acids, more preferably comprises 110 to 150 amino acids, or 120 to 140 amino acids, and is a consensus sequence of PFAM (SEQ ID NO: 3). A sequence comparison with a certain PF00337 has a bit score of at least 150, more preferably at least 175, and even more preferably 200 or more.

  In order to calculate a bit score from a sequence comparison between a specific sequence and PF00337 which is a common sequence of PFAM (SEQ ID NO: 3), the target sequence is searched with HMM (PFAM database, release 2.1) which is a PFAM database. Initial value variables can be obtained at www.sanger.ac.uk/Software/Pfam. A description of the PFAM database can be found in the reference (Sonnhammer et al., Supra), and a detailed description of the HMM can be found in, for example, the references (Gribskov et al., Meth., Enzymol. 183: 146, 1990 and Stultz et al. , Protein Sci. 2: 305, 1993).

  The galactoside binding region of galectin-8 further comprises one, preferably two protein kinase C phosphorylation site (PROSITE number, PS00005), casein kinase II phosphorylation site (PROSITE number PS00006) and / or a characteristic sequence of galaptin. (PROSITE number, PS00309). The protein kinase C phosphorylation site has the following consensus sequence: [ST] -X- [RK]. The characteristic sequence of galaptin is the following consensus sequence: W- [GEK] -X- [EQ] -X- [KRE] -X (3,6)-[PCTF]-[LIVMF]-[NQEGSKV] -X- [GH] -X (3)-[DENKHS]-[LIVMFC]. In certain embodiments, the galactoside binding region of galectin-8 includes the following amino acids and regions of SEQ ID NO: 4, L123-L124, G126, P131, R128, L140-I146 and other sites similar to those previously described. These amino acids and regions are conserved in some mammalian galectins-8 and play a catalytic and structural role (see amino acids labeled with “*” in FIG. 3).

  Certain galectin-8 proteins of the present invention comprise the human galectin-8 amino acid sequences set forth in SEQ ID NOs: 4 and 5. Other galectin-8 of the present invention comprises an amino acid sequence substantially identical to the amino acid sequence set forth in SEQ ID NO: 4 or 5. “Substantially identical” as used herein refers to a first amino acid comprising a sufficient or minimal number of amino acid residues, which amino acid residues are aligned amino acid sequence residues of a second amino acid sequence. , Whereby the first and second amino acid sequences can have a common structural region and / or a common functional activity. For example, at least 60% or 65% identity with SEQ ID NO: 4 or 5, preferably at least 75% identity, more preferably at least 85%, 90%, 91%, 92%, 93%, 94%, The amino acid sequence comprising a common structural region with 95%, 96%, 97%, 98% or 99% identity is made substantially identical to the amino acid sequence of SEQ ID NO: 4 or 5. In particular, proteins containing accidental or intentionally induced mutations such as deletions, additions, substitutions or modifications at specific amino acid residues of SEQ ID NO: 4 or 5 are defined in the galectin-8 protein as described herein. include. It will also be appreciated that the galectin-8 protein may comprise a region represented by the amino acid sequence of galectin-8 of other mammalian species, as defined herein. Other mammalian species include, but are not limited to, cows, dogs, cats, goats, sheep, pigs, mice and horses.

  Dry eye syndrome is considered the “cold” in ophthalmology and is the second most common medical condition found in patients visiting ophthalmologists. Nearly 15 million Americans suffer from dry eyes. The disease is caused by a structural or functional defect in the tear film. The tear film has a dynamic structure, and the production and turnover of the tear film is essential for maintaining a healthy corneal surface. The tear film is composed of three layers: mucin (innermost), water-soluble (intermediate) and lipid (surface) layers. Dysfunction of either of these layers causes dry eyes. The tear film binds to epithelial cells through the innermost mucin layer. The mucin layer is necessary to promote uniform distribution of the tear film throughout the cornea and to properly wet the surface. If mucin molecules do not adhere to the eye, epithelial damage occurs even during normal aqueous tear production. Tear layer diffusion disturbances can interfere with the ocular surface or tears and can cause dry eyes. The innermost mucin layer is produced by conjunctival goblet cells and conjunctival or corneal epithelial cells. Ocular mucins are either transmembrane (eg, MUCI and MUCU) or gel forming / secreting (eg, MUC5AC).

  Mucin is a very large glycoprotein with O-linked carbohydrates in at least half of its mass. Due to the abundant side chains of O-linked carbohydrates, mucin is characterized by being very hydrophilic. This hydrophilic property is believed to allow the water-soluble layer to diffuse uniformly over the eye.

  The present invention is based on the therapeutic use of galectins in the treatment of dry eye syndrome (DES). Without being limited to a particular mechanism, the use is based on the property that galectins bind carbohydrates. Local administration of ophthalmic solution promotes the diffusion of ascitic fluid, which is facilitated by binding of secreted mucins to transmembrane mucins and other glycoproteins at the epithelial surface on the conjunctiva and cornea.

  In the examples herein, three different dry eye mouse models are used to identify the effects of topical administration of galectin solution. In the first model, tear deficiency is determined by the method reported by Pflagfelder's Laboratory, in which a scopolamine patch is administered to the tail of normal mice (Dursun et al., 2002, Invest Ophthamol & Vis Sci. 43: 632-638 And Pflugfelder et al., 2003, The Ocular Surface, 1: 31-36). The second model utilizes MRL / lpr autoimmune mice, which are Sjogren's syndrome (Jabs et al., 1991, Invest Ophthamol Vis Sci. 32: 371-380 and Jabs et al., 1997, Curr Eye Res 16: 909--916), which causes lacrimal inflammatory lesions. In the third model, an infusion of IL-1α is used to create an animal model. The model is the Sjogren syndrome (see Zoukhri, D. et al., Invest Ophthalmol Vis Sci 42 (5): 925-932) model.

  Measurements include tear production by in vivo analysis, tear fluid clearance and corneal immunostaining, and the effects of various concentrations of galectin-1, galectin-3, galectin-7, and galectin-8, either alone or in these parameters. Identified relative to control animals treated in combination and solvent treated. Subsequent to in vivo analysis, biochemical analysis using isolated lacrimal gland and immunohistological analysis of the corneal surface are performed. Furthermore, the effect of pretreatment with a galectin solution performed before scopolamine treatment and the effect presumed by the treatment in combination with galectin and β-lactose which is a galectin inhibitor are also examined.

  According to the data obtained from the examples herein, galectin had a statistically significant effect on measurable parameters in xerophthalmia according to the model. A decrease in corneal immunostaining was seen in the cornea of the galectin-treated animals compared to the placebo-treated animals, providing evidence that galectin has great therapeutic potential for human DES. . Since the two animal models show different types of DES, the positive results obtained with either model can help identify the role of galectins in DES treatment and guide the focus in clinical trials. It becomes. Clinical trials include additional animal experiments to determine the galectin concentration necessary to maximize beneficial effects, the duration of the effect, and the binding properties of the galectin with the cornea and endogenous mucin. Further toxicity tests will be performed to identify safe corneal dose ranges and meet the requirements of the US Food Hygiene Administration before being approved for clinical trials.

The innermost side of the tear is composed of conjunctival goblet cells and mucin produced by the cornea and conjunctival epithelial cells. Mucin is released and exists as secretory mucin, or remains attached to the epithelium as transmembrane mucin. These molecules are glycoproteins with abundant O-linked carbohydrate side chains. Due to the side chain, mucin has a very hydrophilic property, which allows the water-soluble layer to diffuse evenly over the eye. The aqueous layer is produced by lacrimal gland cells and constitutes about 90% of the tear film. The aqueous layer consists mainly of water, in which salts, glucose, lysozyme, tear-specific prealbumin, lactoferrin, secretory immunoglobulin A, and other proteins are dissolved. The outer membrane slows evaporation and consists of sebaceous glands produced by the meibomian glands. The blinking diffuses the layer over the tear film (Rheinstrom SD, 1999, Dry eye. In Yanoff, ed, Ophthalmology. 1 ^st Ed. Editor. Mosley International Ltd, St Louis, MO).

  The National Eye Institute classifies symptoms of dry eyes into two categories: water-soluble layer defects and evaporation defects. However, clinical symptoms are often a mixture of the two pathogenesis (decrease in tear production often leads to the diffusion of the defective oil layer, resulting in over-evaporation. Often associated with hyposensitive dry eye). Water-soluble tear deficiencies are classified into subcategories of Sjogren's Syndrome and Non-Sjogren's Syndrome, and are classified according to the severity of symptoms. Foulks, 2003, The Ocular Surface 1: 20-30).

  Aqueous layer defects are the most common cause of dry eyes, usually due to decreased tear secretion from the lacrimal glands, but also due to increased tear evaporation. Causes of decreased secretion include Shugren's syndrome, senile hypersecretion, lacrimal gland excision, vitamin A deficiency, immune lacrimal gland damage in sarcoids and lymphomas, loss of sensory or motor reflexes, conjunctival scar status, and contact lens wear (Rolando and Zierhut, Surv Ophthlmol. 45: S203-S210, 2001). Changes in composition in the water-soluble layer are also associated with ocular surface damage. Changes in composition include increased electrolyte concentration, loss of growth factors or the presence of inflammatory cytokinins and are associated with ocular surface damage as well as low turnover of tears.

Goblet cell depletion therefore leads to a decrease in mucin content and is associated with many aspects of dry eyes. In particular, some diseases cause goblet cell loss. These include vitamin A deficiency and conjunctiva leaving scars. Vitamin A is essential for maintaining goblet cells and mucin on the ocular surface. Such conjunctiva includes Stevens-Johnson syndrome, trachoma, pemphigoid, and chemical burns. Topical drug administration and preservatives can also damage the ocular surface and goblet cells (Rheinstrom SD, 1999 Dry eye. In Yanoff, ed Ophthalmology. 1 ^st Ed. Editor. Mosley International Ltd, St Louis MO; Abelson et al. ., 2003, Rev Ophthalmol 10: 1).

  In addition to assessing patient discomfort, diagnostic techniques have proven useful in identifying the severity and cause of DES. The tear film instability is assessed by non-invasive tear breaking time measurement (TBUT). In the measurement, the time from when the fluorescent solution is administered and the tear film is first destroyed is observed with a living microscope. Tear production is estimated by a typical or modified skimmer test. The degree of ocular surface staining with fluorescein, or similar dyes, is routinely used to diagnose the severity of ocular surface alternation. These dyes stain the epithelial surface deficient in mucin protein production or the epithelial surface where the epithelial cell membrane is exposed. A standard scoring system has been developed to quantify the severity of injury. The test is routinely performed in clinical trials, and the therapeutic effect of invasive dry eye treatment has been evaluated (Foulks 2003, The Ocular Surface, 1: 20-30).

Artificial tears are the heart of current dry eye treatment. A variety of products are available, but they only temporarily relieve symptoms. At present, there are no treatments that can restore symptoms. Artificial tears can be developed by adding a high molecular weight polymer such as cellulose ester (methyl cellulose, hydroxypropyl methyl cellulose) or polyvinyl alcohol to physiological saline. Other artificial tear formulations are tailored to mimic the mucin composition in tears. Research has shown that the preservatives used in artificial tears have certain toxicities, and no preservatives are contained, and tears in small bottles are available. (Rheinstrom SD, 1999 Dry eye. In Yanoff, ed Ophthalmology. 1 ^st Ed. Editor. Mosley International Ltd, St Louis MO; Abelson et al., 2003, Rev Ophthalmol 10: 1).

  With an improved understanding of the pathophysiology of external diseases, many different modes of treatment have been introduced. These include diseases that target tear lipid membranes and the underlying immune or hormonal pathogenesis (Brewitt et al., 2001, Surv of Ophthalmol 45: S119-S202). For clinical treatment, local secretagogues such as 15 (S) -HETE (hydroxylated eicosatetraenoic acid) (Gamache et al., 2000, Cornea 19: 6: S88) and synthetic P2Y2 receptors Agonists (Jumblatt et al., 1998, Exp Eye Res 67: 341-346) are included, including anti-inflammatory substances such as cyclosporin A and corticosteroids (Pflugfelder, 2003, The Ocular Surface, 1: 31- 36).

  Mucin is known to be responsible for maintaining a healthy physiological function of the ocular tear film. Mucin is a high molecular weight glycoprotein with a protein backbone and high carbohydrate content. In addition to being involved in the mucin layer, the mucin itself forms a sugar coating, ie a scaffold structure, which promotes cell adhesion. Mucins also play a protective role against ocular surface damage. Mucin also plays a role in making the tear film hydrophilic. Thereby, the tear film is stabilized, the corneal tension is reduced, and the water-soluble layer can be uniformly diffused on the ocular surface. Without this mucin layer, tears do not stay on the surface and the surface of the eye is vulnerable (Abelson et al., 2003, Rev Ophthalmol 10: 1).

  The body produces two types of primary mucins, transmembrane and secretory. Transmembrane mucins (MUC1, MUC2 and MUC4) are embedded in the lipid bilayer of cells. Transmembrane mucins are expressed by corneal and conjunctival non-goblet cells and are thought to promote the diffusion of secretory mucins (MUC5AC and MUC7). The secretory mucin is secreted by goblet cells that cross the eye epidermis. The two mucins together form a raw tear film (Danjo et al., 1998, Invest Ophthalmol Vis Sci 39: 2602-2609 and Watanabe 2002, Cornea 21: S17-S22).

Conjunctival mucin deficiency contributes to certain types of dry eye disease. Possible causes include decreased goblet cell density, mucin distribution or changes in properties, and the phenomenon of mucin mRNA expression (Gipson et al., 2000, Prog Retinal Eye Res, 16: 1: 81-98 and Gilbard , 2000, "Dry-eye disorders", In: Albert M et al, eds Principals and Practice of Ophthalmology:.. 2 nd Edition, Philadelphia: WB Saunders Company: 982-1001). Vitamin A deficiency, topical medication, overdose of drops containing preservatives, and scarring conjunctival damage can all lead to goblet cell and ocular surface damage (Danjo et al., 1998, Invest Ophthalmol Vis Sci 39: 2602-2609 and Abelson et al., 2003, Rev Ophthalmol 10: 1).

  Also, transmembrane mucins found on the ocular surface are altered in patients with dry eye. According to researchers, MUC1 and the like, which are transmembrane mucins, are not glycated in dry eye patients, but normal patient mucins are glycated. Structural changes can alter mucin function on the ocular surface, further exacerbating dry eye disease (Danjo et al., 1998, Invest Ophthalmol Vis Sci 39: 2602-2609).

<Role of galectin in tear film dysfunction>
Galectins are carbohydrate binding proteins that have the potential to be ideal candidates for promoting tear film diffusion on the cornea and conjunctival surfaces. These proteins bind mucins with high affinity, in particular the mucin O-linked carbohydrate side chains. Although not bound based on a particular theory or mechanism of action, galectins are found on the cornea and conjunctival surfaces by the tear film by binding secreted mucins to transmembrane mucins (or other glycoproteins). Promote the spread of In mucin-deficient eyes, exogenous galectins extend or promote mucin binding. Furthermore, galectin itself binds to the carbohydrate-binding protein on the corneal surface, thereby further promoting uniform diffusion of the tear film even in the mucin-deficient state.

  Mucin and galectin interactions have been demonstrated in tissues other than the eye. Galectins have been shown to modulate mucin expression and production with strong binding affinities for various mucins. Galectin-3 binds to mucin from human colon cancer cells and regulates the expression of the mucin in a concentration-dependent manner (Bresalier et al., 1996, Cancer Res 56: 4354-4357 and Dudas et al., 2002, Gastroenterology). 118: 1553). In ovarian tumor cells, the mucin-like glycoprotein CA125 binds to galectin-1 (Seelenmeyer et al., 2002, J Cell Sci, 116: 1305-1318).

  Galectins constitute a family of widely distributed carbohydrate binding proteins and are characterized by their affinity for β-galactoside-containing glycans found on many cell surfaces and ECM glycoproteins. In mammals, there is currently a 14-membered galectin family (galectins-1 to -14), defined by structural similarity in the carbohydrate recognition region (CRD). Galectins are soluble proteins. In the cell, galectins are mainly present in the cytoplasm, and galectins-1 and -3 are also confirmed in the nucleus of proliferating cells. Outside the cell, galectins are found on the cell surface and in the ECM. Like certain growth factors (eg, bFGF) and cytokinins (eg, IL-1), galectins do not have typical signal sequences or transmembrane regions and are still not well understood but are It is secreted via a typical pathway. Galectins such as galectin-1, -3, -8 and -9 are found in a wide range of tissues, whereas galectins such as galectin-4, -5 and -6 show tissue specificity. Galectins-1 and -3 are the most studied galectins. Outside the cell, both lectins are thought to be involved in cell-cell and cell-matrix adhesion. The adhesion has been attributed to conjugation with oligosaccharides on the unique isoforms of fibronectin, laminin, vitronectin and integrin. Most of the galectins have two CRDs or exist as dimers and are functionally divalent. Due to its bivalent nature, it can interact with oligosaccharide chains of secretory mucins, which are glycoproteins of the lectin, on the other hand, and transmembrane mucins or oligosaccharide chains of glycoproteins at the apical surface of the cornea Can promote the tear film diffusion on the ocular surface. Several studies have shown that galectin-3 is expressed in mouse and human corneal epidermis. Exogenous galectins-3 and -7 promote recornification of corneal wounds in a mouse animal model and may be involved in corneal epithelial cell trafficking (Cao et al, 2002, J Biol Chem. 277). : 42299-4230 and 2003, Arch Ophthalmol, 121: 82-86).

In the preliminary studies in the examples herein, two different models of corneal wound healing were used, and wound re-epithelialization was previously wild-type in galectin-3 deficient (gal3 ^{− / −} ) mice. It has been found that it is significantly slower than (gal3 ^{+ / +} ) mice. On the other hand, the corneal epithelial wound occlusion rate was not different from that of the galectin-1-deficient mouse and the wild type. Exogenous galectin-3 and galectin-7 promoted re-epithelialization of corneal alkaline burn wounds in a concentration-dependent manner. The effect was suppressed by a competing sugar, ie β-lactose, but not by sucrose, an unrelated disaccharide (Cao et al, 2002, J Biol Chem. 277: 42299-4230 and 2003, Arch Ophthalmol, 121: 82-86).

<Materials and methods>
The following materials and methods are used in the examples throughout this specification.

<Artificial tear solution containing therapeutic concentrations of galectin-1, -3, -7 and -8>
Standard artificial tear solutions are formulated at galectin concentrations based on animal studies. The solution is treated with a buffer, pH is neutral, isotonic and contains a thickener to increase viscosity such as CMC and HMPC. An ideal control solution is made containing all components except galectin. The solution is not intended for storage because preservatives exacerbate epithelial damage. Thus, fresh solutions are prepared for each sample and frozen by the time of administration. Various formulations include a control artificial tear. The control artificial tears were 10 μg / ml of each galectin-1, -3, -7 or -8, or artificial tears containing a combination of pairs, and 20 μg / ml of galectin-1, -3, -7 Or artificial tears containing -8, or a combination of pairs, and further including artificial tears containing 0.1 M β-lactose and disaccharides. The disaccharide acts as a galectin inhibitor.

<DES animal model: tear deficiency induced by scopolamine in normal mice>
Scopolamine is an anticholinergic drug and targets muscarinic cholinergic receptors. Transdermal scopolamine (scop) patches are commonly used in humans for anti-nausea purposes and in the treatment of motion sickness. According to a published study (Dursun et al., 2002, Invest Ophthalmol & Vis Sci. 43: 632-638), scop treatment using this method reduced the tear production to 20% in the control group. Prominent and statistically significant results were obtained. Both fluorescein clearance in tears and carboxyfluorescein uptake in the cornea were both approximately 3-fold. In addition, placing these animals in an air hood increased these parameters more dramatically. Furthermore, in the mice treated with scop + fan hood, the conjunctival goblet cell concentration decreased by more than 90% compared to the control. The reduction in tear production continued for over 24 hours, as controlled by the dermal patch delivery system, allowing continuous drug delivery. On the other hand, with topical atropine administration, tear reduction lasted only a few hours. Re-administration of a new scopolamine patch will last longer.

  In this example, C57BL / 6 mice (Charles River Laboratories, Wilmington, Mass.) Are used. The mice are sex mixed at 6-8 weeks of age. An unanesthetized mouse is caught by hand, and the middle part of the tail of 1 inch is shaved with an electric razor. Cut transcutaneous scop patches (Novartis, Summit, NJ) into four and administer a quarter to the middle of the tail. Patches are re-applied every 48 hours to maintain stable drug delivery. Animals are placed in a blast hood for 1 hour, 3 times a day, daily during the experiment.

<MRL / lpr autoimmune mouse>
In this experiment, MRL / Mp-lpr / lpr (MRL / lpr) mice (Jackson Laboratory, Bar Harbor, ME) are used. The mouse is a similar gene subsystem that develops autoimmune disease and causes lacrimal inflammatory lesions within 16 weeks of age. Based on pathological and histological evaluation, the mouse is a model of human Sjogren's syndrome (Jabs et al., 1991, Invest Ophthamol Vis Sci. 32: 371-380 and 2001, Curr Eye Res. 16: 909-916). As proposed. The mice used in the examples herein are of at least 16 weeks of age and of a sex mixture.

<Measurement of tear production, tear clearance and corneal staining in animals>
Water-soluble analogue production was measured using cotton yarn impregnated with phenol red (Zone-quick, Oasis, Glendora, CA). Hold the thread with tweezers and apply it to the ocular surface within the outer horn for 1 minute. The scale on the cotton thread is used to measure aqueous tear production by how many millimeters of cotton thread is wet.

  Tear crushing time (TBUT) was measured by administering 2 μL of 1% sodium fluorescein as a drop to the ocular surface. The tear film is observed with a gap lamp biomicroscope and the time until the tear film diffuses is recorded.

  Fluorescein staining is performed immediately after TBUT measurement, and 2 μL of 1% sodium fluorescein is used as a drop, which is administered to the ocular surface. Five minutes after administration, fluorescein staining in the cornea is evaluated using a gap lamp biomicroscope. Fluorescein staining is commonly used clinically to determine corneal surface damage. It is said that the corneal surface of xerophthalmia animals is stained with fluorescein, but the normal corneal surface is not stained. The degree of fluorescein staining is described using a standard F1 rating scale similar to that used in dry eye clinical trials.

<Equivalent treatment>
The treatment and measurement plan is designed to measure short-term effects (2 hours after galequen treatment) and long-term effects (up to 14 hours after galequen treatment). Galectins are expected to have a long-term effect on a four-day treatment regimen. A treatment plan, measurement plan or dose change is equivalent to the examples herein if necessary to produce a beneficial effect on the cornea. For example, if a response is seen with 20 μL / mL galectin treatment and no response is seen at 10 μL / mL, an additional example comprises a higher dose (50 μg / mL).

<Galectin-1, -3, galectin-7 and galectin-8 formulation>
The galectin of the present invention is available to those skilled in the art. Available galectins include not only isolated proteins but also proteins produced by genetic recombination or synthesis, and examples include proteins produced by solid phase methods. According to the present invention, a polynucleotide sequence encoding galectin-3, galectin-7 or galectin-8 can be used as a recombinant DNA molecule, which can be used in a suitable host cell. Direct expression. References (Cherayil et al., Supra, Madsen et al., Supra and Hadri et al., Supra) describe in detail the cloning methods for human galectin-1, -3, -7 and -8, respectively. Yes. Nucleotide sequence or function encoding galectin-1, galectin-3, galectin-7, galectin-8 to express biologically active galectin-1, galectin-3, galectin-7, galectin-8 Equivalent sequences are inserted into an appropriate expression vector. The expression vector shall contain elements necessary for transcription and translation of the inserted encoded sequence. Methods that are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding galectin-1, galectin-3, galectin-7 or galectin-8 and appropriate transcriptional or translational controls. Such methods include in vitro genetic recombination techniques, synthetic techniques, and in vivo recombination or genetic recombination. Deletions, additions or substitutions can be introduced using any technique known to those skilled in the art. An example is mutagenesis by PCR. These techniques are described in references (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, 1989 and Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY 1989)). It can be introduced into a variety of expression vectors / host systems to express galectin-1, galectin-3, galectin-7 or galectin-8. These expression vectors / host systems are infected with recombinant bacteriophages, microorganisms such as bacteria transformed with plasmid or cosmid DNA expression vectors, yeast transformed with yeast expression vectors, viral expression vectors (eg baculovirus) Insect cell lines, plant cell lines transformed with various expression vectors (eg cauliflower mosaic virus (CaMV), tobacco mosaic virus (TMV)) or bacterial expression vectors (eg Ti, pBR322 or pET25b plasmid), or animals Cell lines are included, but are not limited. Alternatively, the galectin of the present invention may be synthesized in whole or in part by using a chemical method and galectin-1, galectin-3, galectin-7 or galectin-8 amino acid sequence. For example, peptide synthesis can be performed using various solid phase methods (Roberge et al., Science 269: 202, 1995), and automatic synthesis can also be performed. For example, 431A peptide synthesizer (Applied Biosystems of Foster City, CA) can be used to synthesize peptides according to the instruction manual attached to the device.

<Pharmaceutical composition>
One feature of the present invention is that a pharmaceutical composition is provided. The composition comprises galectin-1, galectin-3, galectin-7 and / or galectin-8, optionally with a pharmaceutically acceptable carrier. In some embodiments, these compositions optionally comprise one or more additional therapeutic agents. In some embodiments, the therapeutic agent or drug is selected from the following group. The drug is a growth factor, anti-inflammatory agent, vasoconstrictor, collagenase inhibitor, topical steroid, matrix metalloproteinase inhibitor, ascorbic acid, angiotensin II, angiotensin III, calreticulin, tetracycline, fibronectin, collagen, thrombosponge Transforming growth factor (TGF), keratinocyte growth factor (KGF), fibrotic eye cell growth factor (FGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), new It consists of vitamin B such as reguulin (NDF), hepatocyte growth factor (HGF) biotin, and nialuronic acid.

  As used herein, a “pharmaceutically acceptable carrier” refers to any and all solvents, diluents or other liquid media, dispersions or suspension acids, surfactants, isotonic agents, Consists of thickening or emulsifying agents, preservatives, solid binders and lubricants. The reference (Remington's Pharmaceutical Sciences Ed. By Gennaro, Mack Publishing, Easton, PA, 1995) discloses a wide variety of carriers used in the formulation of pharmaceutical compositions and describes known methods of dispensing. . Examples of pharmaceutically acceptable carriers include sugars such as glucose and sucrose, starches such as corn starch and potato starch, cellulose such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate and derivatives thereof, powdered tragacanth, malt, Excipients such as gelatin, cocoa butter and suppository wax, oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil, glycols such as propylene glycol, esters such as ethyl oleate and ethyl laurylate Buffer, such as agar, magnesium hydroxide and aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol and phosphate buffer, and also sodium lauryl oxalate and stearic acid magnesium Certain non-toxic compatible lubricants, as well as colorants, release agents, coatings, sweetness, flavoring and flavoring agents, and preservatives and antioxidants can also be included in the composition at the discretion of the formulator. .

<Dose effective for treatment>
Another feature is the treatment of dry eyes by contacting the eye with a pharmaceutical composition according to the treatment method of the present invention and is described below. Accordingly, the present invention provides a method for treating dry eye, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition to a subject in need of treatment. The composition comprises an active ingredient comprising galectin-3, galectin-7 and / or galectin-8, the dosage and frequency of administration being determined according to the desired result. Of course, this includes the process of administering the medicament of the present invention as a therapeutic treatment, which promotes tear film diffusion at the cornea and conjunctival surfaces. Alternatively, administered as a preventive measure to minimize complications associated with dry eyes (eg, as a wound cleaning solution during and / or after surgery, or during and / or after treatment of inflammatory conditions combined with antihistamines) Including the process of In some embodiments of the present invention, a “therapeutically effective amount” of the pharmaceutical composition is an amount effective for dry eye treatment. According to the methods of the present invention, the composition can be administered by any route of administration in any amount effective to promote dry eye treatment. Accordingly, the expression “an amount effective to promote dry eye therapy” herein refers to an amount of the composition sufficient to promote the tear film. The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage amount and dosage are adjusted to provide a sufficient amount of the active ingredient or to maintain the desired effect. Additional factors to consider include severity of symptoms such as degree of dry eyes, history, patient weight, age, sex, dietary habits, time and frequency of administration, concomitant medications, reaction specificity, and Tolerance / response to treatment. Long-acting pharmaceutical compositions can be administered several times daily, daily, for 3 to 4 days, weekly or once every two weeks depending on the half-life and clearance time of the composition.

  The active ingredient of the present invention is preferably formulated as a dosage unit form in consideration of ease of administration and uniformity of administration. The expression “dosage unit form” as used herein refers to a physically independent unit and is a unit of active ingredient appropriate for the patient to be treated. However, the daily usage amount of the composition of the present invention shall be determined by the attending physician within medically reasonable judgment. Effective doses for the treatment of the active ingredient are first estimated using cell culture tests or animal models, generally mice, rabbits, dogs or pigs. Animal models are also used to specify the desired concentration range and route of administration. Although direct administration is considered the route of administration, the information obtained is used to identify useful doses and additional routes of administration for human administration. A therapeutically effective dose refers to the amount of active ingredient that ameliorates symptoms and conditions. The therapeutic efficacy and toxicity of the active ingredient can be identified by standard pharmaceutical techniques using cultured cells or laboratory animals, eg, ED50 (dosage is therapeutically effective in 50% of the population) and LD50 (administration). The amount is fatal in 50% of the population). The ratio between the toxic dose and the therapeutically effective dose is the therapeutic index, for example expressed as the ratio LD50 / ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. Data obtained from cell culture analysis and animal experiments is used in a series of formulations used in humans.

<Administration of pharmaceutical composition>
After being formulated into the desired dosage with a suitable pharmaceutically acceptable carrier, the pharmaceutical composition of the present invention is applied topically to humans and other mammals, such as to the eye (by powder, ointment or infusion), for example to the eye. Direct administration is possible. Alternative and additional routes include oral, rectal, parenteral, intracisternal, intravaginal, intraperitoneal, buccal or nasal routes, and the like and are envisaged depending on the severity of the condition being treated.

  Liquid dosage forms for ophthalmic administration include buffering and solubilizing agents, desired diluents such as water, preservatives such as timosol, and one or more biopolymers or polymers for solution preparation. Examples of the biopolymer or polymer include polyethylene glycol, methyl cellulose hydroxide, sodium hyaluronate, sodium polyacrylate, tamarind rubber, and the like.

  Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to these active ingredients, liquid dosage forms contain inert diluents, and those commonly used in the art include, for example, water or other solvents, solubilizers or emulsifiers {e.g. Ethyl alcohol, isopropyl alcohol, carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil (especially cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil), Glycerol and tetrahydrofurfuryl alcohol, etc.}, polyethylene glycol, sorbitan fatty acid esters, and mixtures thereof. In addition to inert diluents, oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and flavoring agents.

  Topical or transdermal dosage forms of the inventive pharmaceutical composition include ointments, pastes, creams, lotions, gels, powders, aqueous solutions, sprays, inhalants or patches. The active ingredient is mixed aseptically, and a pharmaceutically acceptable carrier and, if necessary, preservatives or buffers are added. For example, eye or skin infections are treated with water-soluble drops, mists, emulsions or creams. Drug administration can be both therapeutic and preventive. Prophylactic prescriptions are applied to potential wounds or causes of wounds and include contact lenses, contact lens cleaning and rinsing solutions, contact lens storage or transport containers, contact lens handling instruments, eye drops, surgical cleaning solutions, ear drops, eye patches and eyes. Used in peripheral cosmetics (including cream, lotion, mascara, eyeliner, eyeshadow, etc.). The present invention includes ophthalmic devices, surgical devices, hearing devices or products (gauze, bandages or strips) comprising the disclosed compositions, as well as methods for making or using these devices or products. These devices may be coated with the disclosed composition, infiltrated, bonded, or otherwise treated with the composition.

  The ointment, paste, cream and gel are not only active ingredients of the present invention, but also excipients such as animal or vegetable fat, oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicon, bentonite , Silicic acid, talc as well as zinc oxide or mixtures thereof.

  Powders and sprays can contain excipients such as talc, silicic acid, aluminum hydroxide, calcium silicate, polyamine powder or mixtures of the drugs in addition to the agent of the present invention. The spray further includes a propellant such as a chlorofluorohydrocarbon.

  Transdermal patches are even better in that they can control the supply of active ingredients to the body. Such dosage forms are made by dissolving or dispensing the compound in the proper solvent. Absorption enhancers can be used to ensure that the compound is well distributed on the skin. The rate can be controlled by using a rate adjusting membrane or by dispersing the compound in a polymer matrix or gel.

  Injectable preparations can be formulated using dispersing or wetting agents and suspending agents, for example, based on the known art of sterile aqueous or oily suspensions. The sterile injection preparation may be a sterile injection, suspension or emulsion containing a nontoxic and parenterally acceptable dilution or solvent (eg, 1,3-butanediol aqueous solution, etc.). Among the acceptable solvents or solvents used are water, Ringer's aqueous solution, U.S.A. S. P. And isotonic sodium chloride solution. In addition, normally sterile, non-volatile oils are used as solvents or suspending agents. For this purpose, any fixed oil can be used including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in injection preparations. Injectable preparations can be sterilized, for example, by sterilization by filtration using a bacteria-retaining filter, or by sterilizing agent in the form of a sterilized solid composition previously mixed by dissolving or diffusing in sterilized water or other sterilized injection solvent. can do. In order to maintain the effect of the active ingredient, it is preferable to delay the absorption of the active ingredient by subcutaneous or intramuscular injection. In order to delay absorption of the active ingredient by parenteral administration, the active ingredient can be dissolved or suspended in an oil solvent. Depot injectable forms are made by forming microcapsules containing the active ingredient in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of active ingredient to polymer and the nature of the polymer used, the percentage of active ingredient released can be controlled. Examples of biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable preparations can also be prepared by entrapping the active ingredient in liposomes or microemulsions, which are compatible with body tissues.

  Rectal or vaginal compositions are preferably suppositories, which can be prepared by mixing the active ingredient of the present invention with a suitable nonirritating excipient or carrier. Such excipients or carriers include cocoa butter, polyethylene glycol or suppository waxes, which are solid at room temperature but become liquid at body temperature, so they dissolve in the rectum or vaginal cavity and release the active ingredient.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In the solid dosage form, the active ingredient comprises at least one inactive and pharmaceutically acceptable excipient or carrier (such as sodium citrate or dicalcium phosphate) and / or
a) Injection or bulking agent (starch, sucrose, glucose, mannitol, silicic acid, etc.)
b) binders (for example, carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, acacia, etc.),
c) a humectant (such as glycerol), d) a disintegrant (such as agar, calcium carbonate, potato or tapioca starch, alginic acid, specific silicic acid and sodium carbonate),
e) Solution dyeing agent (paraffin etc.),
f) Absorption accelerator (quaternary ammonia compound etc.),
g) wetting agents (eg, cetyl alcohol and monostearate glycerol, etc.),
h) Absorbent (such as kaolin and bentonite clay),
i) Lubricant (talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate)
And mixed with these

  A similar solid composition can be used as an injecting agent in the form of a soft or hard gelatin capsule, and an excipient such as lactose as well as high molecular weight polyethylene glycol is used. Solid dosage forms are formulated into tablets, dragees, capsules, pills, and granules using coatings or shells (enteric coatings, controlled release coatings and other coatings known as pharmaceutical formulation techniques). In the solid dosage form, the active ingredient is mixed with at least one inactive diluent such as sucrose or starch. The dosage form can also include additional substances other than non-active diluents as is commonly done, such as tablet lubricants and other tablet adjuvants such as magnesium stearate and microcrystalline cellulose. It is done. In the case of capsules, tablets and pills, the dosage forms can also comprise buffering agents. An opacifier can optionally be included. An opacifier releases an active ingredient lately only in a specific part of the intestinal tract or selectively in a specific part of the intestinal tract. Examples of embedding compositions that can be used include polymeric substances and waxes.

<Use of pharmaceutical composition>
As described in more detail in the description and examples above, galectin-3, galectin-7, and galectin-8 are useful in promoting tear film diffusion, which allows the oligosaccharide chains of secretory mucins to be This is done by binding to a transmembrane mucin (or other glycoprotein). In general, galectins are considered clinically effective in promoting the healing of dry eye.

  In general, galectins are not limited to skin epithelium, any skin epithelial tissue, corneal epithelium, gastrointestinal lining, lung epithelium, and renal canal, vascular epithelium, uterus and vaginal epithelium, urethral epithelium, or respiratory epithelium It has been shown herein to be useful in promoting healing associated with the inner epidermis. The present invention covers a wide variety of embodiments and heals a variety of epithelial wounds. Epithelial wounds include, but are not limited to, surgical wounds, excisional injuries, blisters, ulcers, lesions, detachments, folds, lacerations, burns, wounds, pain, and burns from heat or chemical exposure. . The wound may be of a healthy person, but symptoms that induce abnormal wound healing (diabetes, corneal dystrophy, urethra, malnutrition, vitamin deficiency, obesity, infection, immunosuppression, steroids, radiation It may be the wound of a patient with treatment, complications associated with systemic treatment with non-steroidal anti-inflammatory drugs (NSAIDs), antineoplastic drugs and antimetabolites.

  Galectins-1, -3 and -7 and / or -8 are used herein to help promote skin recovery that occurs after, for example, skin loss. Alternatively, for galectin-1, galectin-3, galectin-7 and / or galectin-8, herein, the adhesion of the skin graft to the wounding layer is enhanced and the wounding layer Again, it was shown to promote epithelialization. Suitable skin grafts include autografts, artificial skin, allografts, autografts, self-epithelial grafts, avascular grafts, Blair-Brown grafts, bone grafts, embryonic tissue grafts, Dermal graft, delayed graft, skin graft, epidermal graft, fascia graft, full-thickness graft, xenograft, allograft, transplantable graft, superficial graft, reticular graft, mucosal graft , Including Ollier-Thiersch grafts, omental grafts, scissor grafts, stalk grafts, penetrating grafts, mid-skin grafts, and split-skin grafts. It is not limited.

  Galectin-1, -3, -7 and / or galectin-8 are herein useful for treating herpetic dermatitis. This herpetic dermatitis forms herpes at the junction of the dermis and epithelium. Galectin-1, -3, -7 and / or galectin-8 are used herein to treat epidermolysis bullosa and to treat a loss of adhesion between the epithelium and the underlying dermis. Useful. In addition, this loss of adhesion frequently occurs. Galectins-1, -3, -7 and / or galectin-8 are useful for treating open and painful blisters. These treatments can be achieved by galectin-1, -3, -7 and / or galectin-8 by promoting re-epithelialization of these lesions. Galectin-1, -3, -7 and / or galectin-8 can further treat pemphigus with loss of adhesion between cells within the epithelium, or adhesion between cells at the junction of the dermis and epithelium. Helps treat joint pemphigus with loss. Galectins-1, -3, -7 and / or galectin-8 are used to treat various ulcers. Such ulcers include, but are not limited to, diabetic (sex) ulcers, skin ulcers, decubitus ulcers, arterial ulcers, and various congestive ulcers.

  The present invention also includes a method for promoting corneal tissue repair. The methods include corneal ulcer, heat, radiation, foam keratitis, corneal ulcer or tear, photorefractive index surgery for myopia correction, foreign and sterile corneal infiltration, acid or alkali (e.g. hydrofluoric acid, Chemical burns from exposure to formic acid, anhydrous ammonia, cement, phenols, or other chemical agents (yellow phosphorus, elemental metals, nitrate hydrocarbons, tar), neurotrophic keratopathy, diabetic keratopathy, Thygeson's punctate cornea Keratitis such as inflammation, viral keratitis (metaherpetic keratitis, herpetic keratitis) and keratitis such as bacterial keratitis, corneal dystrophy such as lattice dystrophy, epithelial basement membrane dystrophy (EBMD) And treating corneal epithelial defects caused by Fuchs endothelial dystrophy.

  By the methods herein, galectin-1, -3, -7 and / or galectin-8 are useful for treating gastrointestinal ulcers, helping the healing of the mucosal lining, and the glandular and duodenal mucosa The inner layer can be regenerated faster. Inflammatory bowel diseases such as Crohn's disease and ulcerative colitis cause destruction of the mucosal surfaces of the small and large intestines, respectively. Thus, galectin-1, -3, -7 and / or galectin-8 can be used by promoting the remodeling of its mucosal surface, thus helping faster healing and inflammatory bowel disease Can be delayed. Galectins-1, -3, -7 and galectin-8 are harmful substances (for example, ingested harmful substances or harmful substances caused by surgery) by immobilizing mucin and binding the mucin to the tip of the epithelium. Can protect the gastrointestinal tract from Galectins-1, -3, -7 and / or galectin-8 are used to reduce the side effects of enterotoxicity. Said enterotoxicity is caused by bacterial infection, viral infection, radiation therapy, chemotherapy or other treatments. Galectin-8, -3 and / or galectin-7 prevent or relieve prophylactically or therapeutically, for example, mucositis, esophagitis or gastritis. (For example, damage associated with oral, esophagus, intestinal tract, colon, rectum, anal ulcer is healed.)

  Galectins-1, -3, -7 and / or galectin-8 are beneficial in promoting urothelial healing. Tissue layers containing urothelial cells are damaged by many means. Such means include catheterization, surgery, or bacterial infection (eg, infection by a mediator that causes a sexually transmitted disease, such as gonorrhea). The invention also encompasses a method for promoting healing of female reproductive tissue. The method comprises administering an effective amount of galectin-8, -3 and / or galectin-7. Damage to the tissues of the female reproductive system is caused by a wide range of diseases including infections such as Candida, Trichomonas, Gardnerella, gonorrhea, chlamydia, mycoplasma and other sexually transmitted diseases.

  Galectins-1, -3, -7 and / or galectin-8 are beneficial by methods that facilitate the treatment of renal epithelial cells. And therefore, it can be beneficial for the relief or treatment of renal diseases and conditions such as acute or chronic renal failure and end-stage renal disease. Galectins-1, -3, -7 and / or galectin-8 are beneficial by methods that facilitate the treatment of breast tissue. And therefore, it may be beneficial to promote healing of breast tissue that has been subjected to surgery, trauma or cancer. Galectins-1, -3, -7 and / or galectin-8 are beneficial by the methods presented herein that promote healing and alleviation of brain tissue damage. The brain tissue damage is due to trauma, surgery or chemicals.

  Galectin-1, -3, -7 and / or galectin-8 can be administered prophylactically to reduce or avoid damage to the lungs caused by various pathological conditions. For example, galectin-1, -3, -7 and / or galectin-8 can facilitate the treatment of alveoli and bronchiole epithelium and avoid, attenuate or treat acute or chronic lung injury. Galectin-1, -3, -7 and / or pulmonary emphysema leading to progressive loss of alveoli, or respiratory tract disturbances, ie smoke inhalation and burns, which cause bronchiolar epithelium and alveolar necrosis Alternatively, galectin-8 can be effectively treated. The galectin-1, -3, -7 and / or galectin-8 can treat injuries resulting from chemotherapy, radiation therapy, lung cancer, asthma, black lung and other lung injury situations.

  Of course, the treatment methods encompassed by the present invention are not limited to the treatment of human wounds and may be used for the treatment of any mammalian wound. Such mammals include but are not limited to cattle, dogs, cats, goats, sheep, pigs, mice and horse species. When treating certain types of wounds, the galectin-1, -3, -7 and / or galectin-8 used may be galectin-1, -3, -7 and / or galectin-8 which occurs naturally in the species. It is preferred to have an amino acid sequence that is substantially identical to the amino acid sequence of, but is not limited thereto.

  All animals mentioned in these examples are in compliance with the recommendations of the Ophthalmology and Vision Research Council on the Use of Animals in Ophthalmic Research, and the Guidelines on NIH Laboratory Animal Care and Use Yes.

<Example 1: Upregulation of galectin-3 in movement of corneal epithelium accompanying injury>
Mice with 2 mm excimer laser excision and excisional wounds that can be partially healed in vivo to determine whether the expression level of galectin-3 changes in the epithelium of the healing cornea with injury Were immunostained with rat anti-human galectin 3-mAb M3 / 38 (American Culture Collection, Rockville, MD). The corneal epithelium is the prototype of the stratified epithelium. In mice, the corneal epithelium comprises 20-25% of the total corneal thickness and consists of 5-6 layers of cells. The lower surface of the epithelial basement membrane is provided with a corneal stroma. In the case of a mouse, the corneal stroma is 70 to 80% of the total cornea thickness. A resected wound removes the epithelium without damaging the corneal stroma. Conversely, excimer laser treatment is commonly used to correct myopia and removes not only the epithelium but also the corneal stroma located on its underside.

  Swiss Webster mice (Taconic Laboratory Animal Service, Germantown, NY) were anesthetized by intramuscular injection with 1.25% avertine (0.2 ml / 10 kg body weight). The avertine was prepared by mixing 2.5 g of 2,2,2 tribromoethanol, 5 ml of 2-methyl-2-butanol (Aldrich, Milwaukee, Wis.) And 195 ml of distilled water. Proparacaine eye drops (ALCAINE® available from Alcon Labs Fort Worth, TX) were used as local anesthesia for the cornea. Transepithelial excimer laser excision was performed on the right eye of group 1 mice using APEX PLUS® Excimer Laser (Summit Technology of Waltham, Mass.) (2 mm optics, depth of excision from 42 to 44 μm). Well, PTK mode). A 2 mm excised wound was made using the Alger brush (Alger Equipment Company of Lago Vista, TX) on the right eye of the second group of mice.

Along with the surgery, all animals were injected intramuscularly using buprenorphine (0.2 ml of 0.3 mg / ml, BUPRENEX® available from Reckitt & Colman Pharmaceuticals, Richmond, VA) as an analgesic. Upon application of antibiotic ointment (VETROPOLYCIN® available from Pharmaderm, Melville, NY), the cornea was partially healed in vivo after 16-18 hours. At the end of the healing period, the animals were anesthetized as described above and then dissected by dislocation of the neck. After dissection, the eyes were fixed with formalin for 2 hours and then embedded in paraffin wax. A tissue fragment (thickness 5 μm) was cut at a position parallel to the eyeball axis. The fragment was deparaffinized by treatment with xylene and rehydration using graded ethanol solutions (100%, 70%, 30%). For immunostaining, tissue fragments were continuously cultured in 3% H ₂ O ₂ (37 ° C., 10 minutes), 2.5% standard goat serum. Respectively, H ₂ O ₂ inhibits endogenous peroxidase activity and goat serum inhibits non-specific binding. The fragment was continuously cultured with mAb M3 / 38 (undiluted hybridoma fluid, 1 hour) and biotinylated with anti-rat immunoglobulin (IgG) for 1 hour (1: 200, Vector Labs, Burlingame, CA). A new complex of avidin D, biotin peroxidase (Vector Lab) and diaminobenzidine (DAB) -H ₂ O ₂ reagent (Kirkegaard & Perry Labs, Gaithersburg, MD) was prepared in 20 hours. As a negative control, fragments were treated with irrelevant mAb or medium alone.

  FIG. 9 shows a standard cornea (FIGS. 9A and B) and a healing cornea (FIGS. 9C and D) in an immunohistochemically stained paraffin fragment. FIG. 9 shows that in both corneal wound healing models, the mobile healing corneal epithelium is stained more strongly with mAb M3 / 38 than the standard epidermis, and in particular, the basal and intermediate cell layers are darker. Shown to be stained. In both the healing corneal epithelium and the standard corneal epithelium, immunostaining is more intensely stained at the cell-matrix adhesion site. Standard corneal stromal cells do not react with mAb M3 / 38, while cells located in the anterior corneal stroma under the healing cornea ensure galectin-3, especially in the region under the migratory epithelium. To express.

  The immunoreactivity of galectin-3 in the corneal epithelium is similar to that when the cornea is cured by organ culture for 16-18 hours in Eagle's minimum essential medium without serum. The Eagle's minimum essential medium contains non-essential amino acids, L-glutamine, antibiotics, 0.4% bovine serum albumin (BSA). However, the anterior corneal stroma is healed in vivo without cells that express galectin-3, so that galectin-3 positive cells found in the corneal stroma that may heal in vivo are kerato It was suggested that it was more likely to be leukocytes than sites.

In order to determine whether the carbohydrate recognition region of galectin-3 is involved in corneal epithelial layer migration following injury, the cornea with 2 mm excimer laser ablation and excision wounds were treated with the disaccharides β-lactose and sucrose. Healed by organ culture in the presence or absence of. β-Lactose contains galactose and binds to galectin, while sucrose has no galactose and does not bind to galectin. According to these experiments, the reepithelialization rate of corneal wounds was significantly slow in the presence of β-lactose, while sucrose had no effect. As shown in FIG. 10, the cure rate between different groups (mean ± standard error from at least two experiments) is expressed in mm ² / h as follows: 0.088 ± 0.003 (N = 29) for the medium alone, 0.063 ± 0.003 (N = 19) for the medium and β-lactose, and 0.084 ± 0.004 (N = 10) for the medium and sucrose. )Met.

<Example 2: Occlusion of corneal epithelial wound in wild type mouse and galectin-3 deficient mouse>
Four different models of corneal wound healing were used to determine whether corneal wound re-epithelialization was reduced in galectin-3 deficient mice. Galectin-3 deficient mice (gal-3 ^-/- ) were produced for the blockade of the galectin-3 gene described in Hsu et al., Am J. Pathol 156: 1073,2000. In particular, the region encoding CRD was blocked by the neomycin resistance gene. This involves replacing 5 fragments of the 0.5 kb intron 4-exon with an antibiotic resistance gene (neo). Inactivated galectin-3 gene was confirmed by Southern blotting and Western blotting.

Briefly, excimer laser ablated corneas (as described in Example 1) or alkali burns are partially healed in vivo or in vitro (as described in Example 1). It was. For alkali damage, a 2 mm filter disk (Whatman 50, Whatman International, Maidstone, UK) was made by using trephine and soaked in 0.5N sodium hydroxide, which was then applied to the right eye of the second group of mice. It was placed on the cornea surface for 30 seconds. The eyes were then washed with sufficient PBS. At the end of the healing period, the wound area was visualized by staining with methylene blue. Thereafter, the stained wound was photographed at a standard distance, and the outline of the wound area was copied on paper from the image of the stained wound. These contours were digitized and quantified using SIGMASCAN® software (SPSS Science of Chicago, IL). In analysis of wound occlusion rates in gal-3 ^{+ / +} mice of different models of wound healing, the wound occlusion rate, expressed as gal-3 ^{+ / +} mm ² / h, is more alkaline in the cornea damaged by Excimer laser. It was found to be lower than the cornea damaged by burns caused by. Regardless of the method of injury, the wound occlusion rate is higher in the cornea that heals in vivo than in the cornea that heals in organ culture. As shown in FIG. 11, the wound occlusion rate between the gal-3 ^{+ / +} groups was 0.076 ± 0.003 mm ² / h in the excimer laser / in vivo group, and 0.050 ± in the exima laser / in vitro group. 0.003 mm ² / h, burns due to alkali / in vivo group is 0.182 ± 0.003 mm ² / h, burns due to alkali / in vitro group is 0.106 ± 0.005 mm ² / h. Values for each group are shown as the mean ± standard error of at least two experiments (each group N = 9 or more). Comparing the wound occlusion rates of the gal-3 ^{+ / +} and gal-3 ^{− / −} groups, regardless of whether the cornea was damaged by excimer laser or alkali treatment, Regardless of whether healed in vitro, the corneal epithelial wound occlusion rate expressed in mm ² / h is significantly higher in mice in the gal-3 ^{− / −} group than in mice in the gal-3 ^{+ / +} group It became clear that it was low. Different gal-3 ^{- / -} wound closure rate in the group is Aix Shima laser / vivo group 0.060 ± 0.004 mm ² / h, Ek Shima laser / vitro group 0.036 ± 0.005 mm ² / h, The alkali burn / in vivo group is 0.150 ± 0.008 mm ² , and the alkali burn / in vitro group is 0.081 ± 0.004 mm ² / h. Again, all values are shown as the mean ± standard error of at least two experiments (each group N = 8 or greater).

<Example 3: Expression pattern of genes in corneal epithelial migration of galectin-3 deficient mice accompanying damage>
In an attempt to understand why re-epithelialization of corneal epithelial wounds is disrupted in gal-3 ^{− / −} mice, the gene expression pattern in gal-3 ^{+ / +} and gal-3 ^{− / −} corneal healing was expressed as cDNA. The results were further confirmed by semi-quantitative RT-PCR. Transepithelial excimer laser ablation (2 mm radius) was made in the right eye of gal-3 ^{+ / +} and gal-3 ^{− / −} mice (as in Example 1). The cornea was partially healed in vivo in 20-24 hours. At the end of the healing period, the animals were dissected, the corneas removed and immediately placed in liquid nitrogen and shipped to Clontech Labolatories, Palo Alto, CA for gene expression analysis using SMART® cDNA technology. . Briefly, total RNA was isolated using reagents defined in the ATLAS® pure total RNA labeling system. The RNA produced from 30 gal-3 ^{+ / +} and gal-3 ^{− / −} corneas was 3.5 μg and 2.6 μg, respectively. The A260: A280 ratios for corneal RNA production of gal-3 ^{+ / +} and gal-3 ^{− / −} mice were 1.48 and 1.37, respectively. The 28S: 18S ratio of ribosomal RNA was 1.8 in both preparations. This confirmed that the RNA production was sufficient. For probe generation, the first helical cDNA was synthesized using 175 ng RNA, modified oligo (dT) primer (CDS primer), POWERSCRIPT® reverse transcriptase, and SMART® II oligonucleotide. did. The control group cultured the sample without reverse transcriptase. CDNA was amplified by long distance (LD) -PCR. To determine the optimal number of amplification cycles, aliquots of reagent products were collected at 15, 18, 21, 24 cycles and analyzed by agarose gel electrophoresis. The yield of double helix cDNA amplified using an optimal number of amplification cycles (eg, 23 cycles) was 1-1.6 μg. The amplified cDNA (500 ng) was radioisotoped using Klenow enzyme and ³³ P-αATP using the SMART® cDNA probe synthesis instructions for the ATLAS® microarray (Clontech). Labeled. The labeled probe was purified by filtration through a NUCLEOSPIN® filter and hybridized with a mouse 1.2k-I ATLAS® nylon cDNA microarray (Clonetech). This is a broad spectrum array of up to 1200 mouse genes. Following hybridization, the membrane was exposed to the screen of a radiation image analyzer and the results were analyzed with ATLAS IMAGE® 2.0 software (Clonetech). Data was confirmed by semi-quantitative RT-PCR.

For RT-PCR, total RNA and first helical structure cDNA were prepared with gal-3 ^{+ / +} healing cornea and gal-3 ^{− / −} healing cornea according to the procedure described above. PCR amplification was performed in a volume of 50 μl using 14 ng of cDNA, gene-specific special primers purchased from Clonetech, and other reagents from ADVANTAGE® 2 PCR kit (Clonetech). The annealing temperature was 68 ° C. and the reagents followed changes in the number of cycles of PCR amplification. For housekeeping gene analysis, an aliquot of 5 μl of amplification product was collected every 5 cycles and at the beginning of 18 cycles, while amplification products of different expressed gene reactions were collected every other cycle and at the start of 28 cycles. Amplified products collected at various cycles were analyzed by electrophoresis on a 1.5% agarose / ethidium bromide gel (FIG. 12). From these experiments, compared to the healing cornea of gal-3 ^{+ / +} mice, the healing cornea of gal-3 ^{− / −} mice showed that galectin-7 mRNA transcripts, other galactose binding proteins and toroid-like proteins (TLL) ), It was found that the amount of metalloproteinase was significantly less. Overall, compared to the gal-3 ^{+ / +} healing cornea, the gal-3 ^-/- healing cornea was about 1/12 of galectin-7 and about 1/14 of the TLL gene transcript (data (Not shown). The expression levels of mRNA transcripts of various housekeeping genes were measured using microarray technology (Fig. 12) and semi-quantitative RT-PCR (Fig. 12, GAPDH was D-glyceraldehyde-3-phosphate dehydrogenase, RPS29 was ribosome. Protein S29, ODC is ornithine decarboxylase) and both gal-3 ^{+ / +} healing and gal-3 ^{− / −} healing were similar.

In order to confirm whether the expression level of galectin-7 protein is also decreased in the healing cornea of gal-3 ^{− / −} mice, the surface activity of the healing cornea of gal-3 ^{+ / +} and gal-3 ^{− / −} Western blot analysis using a drug extract (FIG. 13A) and immunohistochemicals of a polyclonal antibody of anti-galectin-7 using paraffin fragments derived from corneas of gal-3 ^{+ / +} and gal-3 ^{− / −} mice A study (Figure 13B) was conducted. The immune activity was graded as intense (++++), moderate (++), weak (+), or negative (−). It was found that the migration of gal-3 ^{− / −} healing corneal epithelium is significantly less immunoreactive for galectin-7 compared to the migration of gal-3 ^{+ / +} healing corneal epithelium (gal-3 ^{+ / +} : ++++ 36/42, ++ 5/42, +1/42 or less, gal-3 ^{− / −} : ++ 3/42, ++ 26/42, +13/42 or less). In addition, gal-3 ^{− / −} mouse fetal fibroblasts (MEF) grown in cell culture showed a decreased amount compared to gal-3 ^{+ / +} MEF culture (FIG. 13C).

<Example 4: Exogenous galectin-3 promotes re-epithelialization of corneal wound in wild-type mice and galectin-3-deficient mice>
The rate of corneal epithelial wound closure varied in galectin-3 deficient mice (gal-3 ^{− / −} mice) (Example 2), but exogenous galectin promotes epithelial remodeling to treat cornea in organ culture The point of no is interesting. In this study, corneas of gal-3 ^{+ / +} and gal-3 ^{− / −} mice that had been burned with alkali were cultured in serum-free medium. This culturing is performed in the presence and absence of various amounts of recombinant galectin-3.

As described above (Yang et al., Biochemistry 37: 4086,1998), recombinant full-length human galectin-3 was produced and purified in E. coli. Alkaline burn wounds (2 mm diameter) were created in both eyes of anesthetized mice using filter discs impregnated with alkali as described in Example 2. After this wound, the mice were killed, the eyeballs were removed, and the eyeballs were cultured for 18 to 20 hours in the presence or absence of exogenous galectin-3.
The left eye of the mouse is used as a subject, but was cultured only in serum-free medium. The right eye was cultured in serum-free medium containing various test reagents. The test reagents include the following.
(I) Galectin-3 (5 μg / mL to 20 μg / mL)
(Ii) Galectin-3 (10 μg / mL) and 0.1 M β-lactose (iii) Galectin-3 (10 μg / mL) and 0.1 M sucrose (iv) 0.1 M β-lactose (v) 0.1 M sucrose At the end of the healing period, the remaining wound area was stained, imaged and quantified using SIGMASCAN® software (SPSS Science, Chicago, Ill.) As described in Example 2. Each group contained a minimum of 3 eyes and all experiments were performed at least twice.

Exogenous galectin-3 did not alter any rate of re-epithelialization of corneal wounds in gal-3 ^{− / −} mice (see FIG. 14A). However, exogenous galectins promoted the rate of wound closure in gal-3 ^{+ / +} mice. The wound occlusion rate depends on the concentration of 10 μg / mL to 20 μg / mL (0.090 ± 0.010 mm ² / h at 0 and 5 μg / mL (average value of at least 2 experiments ± standard deviation: number of samples) Is 0.129 ± 0.010 mm ² / h (average value of at least two experiments ± standard deviation: the number of samples is 7 or more) at 10 μg / mL, and 0.154 ± 0.004 mm at 20 μg / mL. ² / h (mean value of at least two experiments ± standard deviation: the number of samples is 7 or more).
As shown in FIG. 15, the promoting effect of galectin-3 on corneal epithelial wound occlusion in gal-3 ^{+ / +} mice was specifically suppressed by β-lactose but not by sucrose (10 μg / mL galectin− 3 is 0.127 ± 0.010 mm ² / h (average value of at least two experiments ± standard deviation: the number of samples is 7 or more), and galectin-3 (10 μg / mL) and 0.1M β-lactose On the other hand, it is 0.103 ± 0.014 mm ² / h (average value of at least two experiments ± standard deviation: the number of samples is 7 or more), 0.130 for galectin-3 (10 μg / mL) and 0.1 M sucrose. ± 0.003 mm ² / h (average value of at least two experiments ± standard deviation: the number of samples is 7 or more).

<Example 5: Exogenous galectin-7 promotes re-epithelialization of corneal wounds in wild type mice and galectin-3 deficient mice>
In a separate study, a cDNA microarray (eg, as shown in Example 3) was used to compare gene expression patterns of normal and healing corneas in gal-3 ^{+ / +} mice, It was revealed that the expression of galectin-7 was significantly improved. In connection with the study described in Example 3, these findings indicate that galectin-7 expression is reduced in the healing cornea of gal-3 ^{− / −} mice. Due to this, these findings have led to experimental design to determine whether exogenous galectin-7 promotes healing epithelial remodeling in organ culture.
In this study, corneas of gal-3 ^{− / −} mice bearing alkaline burn wounds were cultured in serum-free medium. This culture is performed in the presence and absence of various amounts of recombinant galectin-7.

By cloning the cDNA (available as an EST clone from the American Type Culture Collection, Manassas, VA) into the pET25b plasmid (available from Novagen, Madison, WI) into E. coli. Recombinant full-length human galectin-7 was created.
Alkaline burn wounds (2 mm diameter) were created in both anesthetized mice using filter discs impregnated with alkali as described in Example 2. After this wound, the mice were killed, the eyeballs were removed, and eyeball cultures were performed for 18-20 hours in the presence or absence of exogenous galectin-7.
The left eye of the mouse is used as a subject, but was cultured only in serum-free medium. The right eye was cultured in serum-free medium containing various test reagents. The test reagents include the following.
(I) Galectin-7 (20 μg / mL)
(Ii) Galectin-7 (20 μg / mL) and 0.1 M β-Lactose (iii) Galectin-7 (20 μg / mL) and 0.1 M sucrose At the end of the healing period, as described in Example 2, SIGMASCAN ( Residual wound area was stained, imaged and quantified using registered trademark software (SPSS Science, Chicago, Ill.). Each group contained a minimum of 6 eyes and all experiments were performed at least twice.

As shown in FIG. 16, exogenous galectin-7 promoted the wound occlusion rate (in the case of medium alone, 0.036 ± 0.006 mm ² / h (average value of at least two experiments ± standard deviation: the number of samples was In the case of galectin-7 (20 μg / mL), it is 0.072 ± 0.004 mm ² / h (average value of at least two experiments ± standard deviation: the number of samples is 10 or more).
As shown in FIG. 16, the promoting effect of galectin-7 on corneal epithelial wound occlusion was particularly suppressed by β-lactose but not by sucrose (0.072 ± for 20 μg / mL galectin-7). 0.004 mm ² / h (average value of at least 2 experiments ± standard deviation: number of samples is 9 or more), 0.050 ± 0.004 mm for galectin-7 (20 μg / mL) and 0.1M β-lactose ² / h (mean value of at least two experiments ± standard deviation: the number of samples is 9 or more), 0.079 ± 0.007 mm ² / h for galectin-7 (20 μg / mL) and 0.1M sucrose ( The average value of at least two experiments ± standard deviation: the number of samples is 9 or more).
As shown in FIG. 16, the rate of injury occlusion was further increased when exogenous galectin-7 was added to the damaged cornea of gal-3 ^{+ / +} mice instead of gal-3 ^{− / −} mice (0.094). ± 0.003 gal-3 ^{+ / +} mm ² / h).

<Example 6: Skin epithelial wound occlusion in wild type mouse and galectin-3 deficient mouse>
Gal-3 ^{+ / +} and gal-3 ^{− / −} mice were anesthetized by intraperitoneal administration of 1.25% tribromoethanol (Avertin) (0.2 mL / 10 g body weight). Prior to the laser treatment, the hair in the back area was shaved using a razor blade. A 6 mm dorsal skin wound penetrating the epithelium was made using an excimer laser (Summit Technology, Warsham, Mass.). After the surgical procedure, antibacterial ointment is applied to the wound surface and bunorphine (2 mg / kg body weight) is administered subcutaneously to minimize postoperative pain. The wound can be partially healed in vivo and is being tested 24, 48 and 72 hours after surgery.
At the end of the healing period, mice are anesthetized again by intraperitoneal administration of 1.25% tribromoethanol (Avertin) (0.2 mL / 10 g body weight), imaged using Sigma Scan software, and then quantified.
Wound occlusion rates are compared between two groups of experimental animals (ie, gal-3 ^{+ / +} mice and gal-3 ^{− / −} mice). The experimental animals are subsequently killed by carbon dioxide inhalation or pentobarbital overdose.

<Example 7: Effect of exogenous galectin-3 on epithelial remodeling of skin wound>
Experimental animals (mouse: 57BL / 6 and 129 mixed genetic background; age: 6 to 8 weeks; sex: mixed) were anesthetized by intraperitoneal administration of 1.25% tribromoethanol (Avertin) (0.2mL / 10g body weight) It was done.
Prior to the laser treatment, the hair in the back area was shaved using a razor blade. Two 6 mm dorsal wounds (one wound on each side) penetrating the epithelium were made using an excimer laser (Summit Technology, Warsham, Mass.).
After the surgical procedure, antibacterial ointment is applied to the wound surface and bunorphine (2 mg / kg body weight) is administered subcutaneously to minimize postoperative pain. The wound was partially healable in vivo, and ointment containing galectin-3 was applied to the right wound and every carrier was applied to the left wound every 4 to 6 hours after surgery. . The left wound plays a role as a target.
At the end of the healing period (24 to 48 hours later), experimental animals were anesthetized by intraperitoneal administration of 1.25% tribromoethanol (Avertin) (0.2 mL / 10 g body weight) and imaged using Sigma Scan software And then quantified.
Wound occlusion rates are compared between two groups of experimental animals (ie, those treated with galectin-3 and subjects). The experimental animals are subsequently killed by carbon dioxide inhalation or pentobarbital overdose.

<Example 8: Effect of exogenous galectin-7 on epithelial remodeling of skin wound>
Experimental animals (mouse: 57BL / 6 and 129 mixed genetic background; age: 6 to 8 weeks; sex: mixed) were anesthetized by intraperitoneal administration of 1.25% tribromoethanol (Avertin) (0.2mL / 10g body weight) It was done.
Prior to the laser treatment, the hair in the back area was shaved using a razor blade. Two 6 mm dorsal wounds (one wound on each side) penetrating the epithelium were made using an excimer laser (Summit Technology, Warsham, Mass.).
After the surgical procedure, antibacterial ointment is applied to the wound surface and bunorphine (2 mg / kg body weight) is administered subcutaneously to minimize postoperative pain. The wound was partially healable in vivo, and ointment containing galectin-7 was applied to the right wound and every carrier was applied to the left wound every 4 to 6 hours after surgery. . The left wound plays a role as a target.
At the end of the healing period (24 to 48 hours later), experimental animals were anesthetized by intraperitoneal administration of 1.25% tribromoethanol (Avertin) (0.2 mL / 10 g body weight) and imaged using Sigma Scan software And then quantified.
Wound occlusion rates are compared between two groups of experimental animals (ie, those treated with galectin-7 and subjects). The experimental animals are subsequently killed by carbon dioxide inhalation or pentobarbital overdose.

<Example 9: Effect of exogenous galectin-8 or galectin-1 on dry eye symptoms in albino rabbits>
Experimental animals (rabbit: albino; age: 6 to 8 weeks; sex: mixed) were anesthetized by intraperitoneal administration of 1.25% tribromoethanol (Avertin) (0.2 mL / 10 g body weight). Daily repeated instillation of 1.0% atropine sulfate (Burgalassi, S., et al., Ophthalm Res 31: 229-235, 1997) causes dry eye symptoms in each rabbit. Evaluation of dry eye syndrome is made for each experimental animal by Schirmer I test and cornea test after fluorescent staining.

Every 4 to 6 hours, an eye drop containing a solution of galectin-8 is administered to the right eye of each experimental animal. Only the carrier is administered to the left eye. The left eye plays a role as a target.
At the end of the healing period (24 to 48 hours later), the experimental animals were anesthetized by intraperitoneal administration of 1.25% tribromoethanol (Avertin) (0.2 mL / 10 g body weight) and the ocular area was replaced with Sigma Scan software. Used to image and then quantified.
Ocular surface and dry eye symptoms are evaluated between two groups of eyes (ie, the right eye treated with galectin-8 and the left eye of interest) and the surfaces are compared.
The experimental animals were subsequently killed by carbon dioxide inhalation or pentobarbital overdose and analyzed according to standard toxicological criteria.

  The amino acid sequences of the various galectin-8 proteins (FIG. 18 and SEQ ID NOs: 4 and 5) of humans and other vertebrates (see FIG. 20) are nearly identical, and this applies to mammals of other species. . Similarly, the amino acid sequences of various galectin-1 proteins (FIG. 19 and SEQ ID NO: 6) of human and other mammals are almost identical. Conserved sites and sites with few conserved changes yield residues that yield other functional galectin-8 and / or functional galectin-1 proteins that treat dry eye symptoms and other ocular symptoms Have the ability.

<Example 10: Scopolamine model: Effect of galectin treatment on DES and pretreatment using galectin solution before scopolamine treatment>
A tear solution containing either 1 or 2 of Galectin-1, Galectin-3, Galectin-7 and Galectin-8 in an amount of either 0, 10 or 20 μg per mL of solution is on the treatment schedule shown below. To be administered.
Treatment for one eye of each experimental animal is performed at each time point with a drop volume of 10 μL. For groups of scopolamine patch models, a 10 μg / mL galectin solution is administered to one eye four times per day. This administration is started immediately after use of the scopolamine patch.
A treatment group consisting of mice is used. Treatment groups and eyes are randomized and coded and measurements are taken randomly. One eye of each experimental animal is administered a treatment solution and the other eye is administered a subject drop. Measurements are made at each point in time.

<Example 11: Scopolamine model: mixed therapy of galectin administered together with β-lactose and galectin inhibitor>
The same procedure as shown in Example 1 was performed. In addition, a 10 μg / mL galectin solution is used together with 0.1 M β-lactose, and it is determined whether or not the effect of galectin is suppressed by saccharides to compete.

<Example 12: Role of galectin-1, galectin-3, galectin-7 and galectin-8 in DES: autoimmune MRL / lpr staining of mice>
Treatment groups and eyes are randomized and coded and measurements are taken randomly. One eye of each experimental animal is administered a treatment solution and the other eye is administered a subject drop. Measurements are made at each point in time.

<Example 13: Treatment effect of galectin-3 on a subject having dry eye symptoms>
To determine the effect of galectin on dry eye symptoms, an animal model system using interleukin-1 was used and four groups of mice were used. Each group consisted of 5 experimental animals and was treated as follows.
Interleukin-1 (IL-1) was injected into the subject on day 0 for the three groups. This injection was performed according to standard procedures created for a mouse model of Sjogren's syndrome (Zoukhri, D. et al., Invest Ophthalmol Vis Sci 42 (5): 925-932). The subject has not received the injection and has not been further treated with eye drops. Subjects who received an IL-1 infusion then received treatment by administering eye drops. This eye drop contains galectin-3, the eye drop amount is 75 μg / mL or 150 μg / mL, and four eye drops are performed per day. This instillation is performed from the first day. One subject group was similarly dosed with buffer only (no galectin) four times per day. The degree of dry eye was assessed for each subject by fluorescein clearance as described above (Zoukhri et al.).

  In the first stage of treatment on the first day, the lacrimal fluid was measured by fluorescein clearance and treated with 75 μg / mL or 150 μg / mL doses of galectin-3, respectively. Compared with the improvement. By day 2, the symptoms of subjects treated with galectin were completely improved. In fact, on the second day, somewhat better tear clearance was observed in subjects with a low dose of 75 μg / mL, indicating that a lower dose may be the optimal dose. Has been. In FIG. 21, the asterisk on the bar of the bar graph (relative to the treated subject on day 2) is from the subject group of subjects with dry eye symptoms who received buffer excipients only. These data are shown to be statistically significant (p <0.05). In conclusion, it can be said that galectin-3 has the ability to treat dry eye and improve this condition within a treatment period of 1 to 2 days. Galectin-1, Galectin-3, Galectin-7 and Galectin-8 are due to the high degree of identity between mammals and the presence of galectin function identity and specific amino acid consensus sequence identity between different types of the galectin family. Any of these or a combination of these can be expected to be effective in the treatment of dry eye and related symptoms as well.

<Conclusion>
Herein, it has been demonstrated that galectin-3 and galectin-7 play a role in re-epithelialization of corneal wounds. In Example 1, immunohistochemistry studies revealed that galectin-3 was present at high density at the intercellular substrate adhesion site of the corneal epithelium after injury. It influences cell-matrix interactions and is an ideal site for cell migration. In Example 2, it was shown that the progress of re-epithelialization of the corneal wound was significantly delayed in the galectin-3 deficient mice than in the wild type mice. In Example 3, it was confirmed that the expression of galectin-7 after injury was significantly reduced in galectin-3 deficient mice than in wild type mice. In Examples 4 and 5, exogenous recombinant galectin-3 and galectin-7 were shown to stimulate corneal wound reepithelialization in gal3 + / + mice. In Examples 6-8, a means for measuring the effect of galectin on the re-epithelialization of the wound was specified. In Examples 9-12, a means for measuring the effect of galectin in dry eye syndrome was clarified. Example 13 showed immediate effect treatment of galectin-3 on dry eyes. Furthermore, in Example 1, it was demonstrated that the stimulatory effect of galectin-3 on the corneal epithelial wound occlusion rate is suppressed by a competitive disaccharide (β-lactose) having a galactoside chemical structure. At the same time, it has been shown that it is not affected by scientifically unrelated disaccharides (sucrose). As a result, it was shown that the sugar chain recognition site is directly involved in the useful effect of exogenous galectin on the cornea.

  With regard to the mechanism by which galectins 1, 3, 7 and galectin 8 affect the treatment of dry eye syndrome, the following is proposed, although not intended to be tied to any particular theory.

As described above, galectin-3 is thought to mediate interactions between cells or cell substrates by binding to complementary complex homogenous including sugar chain polylactosamine. Glycopolylactosamine is found in many extracellular matrices (ECM) or in cell surface molecules such as isoforms of fibronectin, laminin, and integrins. (Liu, Clin. Immunol. 97:79, 2000 and Perillo, supra) However, the findings herein (see Example 4) that exogenous galectin-3 does not promote wound re-epithelialization in gal3 ^{− / −} mice. ) Suggests that intracellular galectin-3 contributes significantly in the process of wound healing. Intracellular galectin-3 has the greatest effect on the expression of specific cell surfaces and / or extracellular matrix (ECM) receptors, and as a result also affects cellular matrix interactions and cell migration. Giving. This idea is consistent with a previously published study in which galectin-3 was stably overexpressed in breast cancer cell lines. The parent cell line expressed little or no galectin. As a result, α4β7, α6β1 integrin increased, and adhesion to various extracellular matrix (ECM) molecules including laminin, fibronectin, and vitronectin became stronger. (Warfield, supra and Mattarese, supra) In another study (Dudas et al, Gastroenterology 118: 1553, 2000) a colon cancer cell line transfected with expressed galectin-3 is the main binding group of the lectin body Increased specific mucin, MUC2. (Bresalier et al, Cancer Research 56: 4354,1996) The fact that the stimulatory effect of exogenous galectin-3 on wound reepithelialization rate in gal3 ^{+ / +} mice is lactose inhibition suggests the following interesting possibilities: doing. Intracellular galectin-3 may actually control the addition of protein sugar chains that function as the extracellular matrix (ECM) receptor of the lectin body and the cell surface. The fact that intracellular galectin-3 acts on the nuclear substrate and has the potential to affect complex biological processes also suggests the following findings. Under certain circumstances, lectins can bind to ribonucleoprotein complexes in the nucleus or act as splicing factors for RNA precursors. (Dagher et al., Proc. Natl. Acad. ScL USA 92: 1213, 1995). Similarly, by Wang et al., Galectin-3 binds to the nuclear matrix in prostate cancer cells and is also single-stranded. It has been demonstrated to be associated with both DNA and RNA. (Wang et al., Biochem. Biophys. Res. Commun. 217: 292, 1995).

By analyzing the gene expression pattern of corneal healing in gal3 ^{+ / +} and gal3 ^{− / −} mice using a complementary DNA microarray of mice, the corneal healing of expressed gal3 ^{− / −} mice was more responsive to galectin-7 than wild type mice. It became clear that it decreased significantly. (See Examples 3, 5) Galectin-7 was first reported in 1994 (Barondes, supra) and is not as well characterized as galectin-3. Unlike galectin-3, galectin-7 has significant tissue specificity. In adult animals, the expression of galectin-7 is limited to the epithelium, and the epithelium is stratified or stratified. (Timmons et al., Supra). Proteins are thought to be involved in cell matrix and cell-cell interactions and apoptosis. (Leonidas, Biochemistry 37: 13930, 1998 and Bernerd et al., Proc. Natl. Acad. Sci. USA 96: 11329, 1999). In general, there is an inverse correlation between galectin-7 expression and keratinocyte proliferation Galectin-7 expression is suppressed by keratinocytes transformed into SV40 virus, and is also suppressed by cell lines derived from epidermal tumors. Exogenous Galectin-3 GAL3 ^{- / -} does not stimulate wound re-epithelialization in the cornea, gal3 ^{- / -} healing of the cornea reduce the amount of galectin-7. This finding suggests that galectin-3 modulates galectin-7 to at least partially affect wound reepithelialization. Indeed, unlike galectin-3, galectin-7 has been found to inhibit lactose and promote wound re-epithelialization at the gal3 ^{− / −} cornea. In addition, gal3 ^{− / −} mouse embryonic fibroblasts were expressed to reduce the amount of galectin-7.

Regardless of the mechanism involved, the finding that both galectin-3 and galectin-7 stimulate re-epithelialization of corneal wounds has broad implications for the treatment of epithelial wounds and especially for non-healing of epithelial wounds. Today, the treatment of persistent epithelial defects in the cornea is a major clinical problem. Furthermore, there is a growing need for effective treatment of post-transplant wounds, elderly chronic wounds, pressure sores, and venous stasis ulcers of the skin. Numerous growth factors known to stimulate cell proliferation (eg, epidermal growth cell factor (EGF), transforming growth factor (TGF), fibroblast growth factor (FGF), KGF, hepatocyte growth factor (HGF)) Has been tested for usefulness in the cornea and has resulted in disappointing overall results in epithelial wound healing of the skin. (Eaglstein, Surg. Clin. North Am. 77: 689, 1997; Singer and Clark, N. Engl. J. Med. 341: 738, 1999; Zieske and Gipson, pp. 364-372 in "Principle and Practice of Ophthalmology Ed. By DM Albert and FA Jakobiec, WB Saunders Company, Philadelphia, PA, 2000; Schultz et al., Eye 8: 184, 1994; Kandarakiset al., Am. J. Ophthalmol. 98: 411, 1984; and Singh And Foster, Am. J. Ophthalmol. 103: 802, 1987). In most of the above studies using growth factors, the acceleration range of wound reepithelialization was significantly smaller than the range identified by galectins in today's studies. . Also, the corneal epithelium treated as a growth factor such as epidermal growth factor (EGF) is hyperplastic (Singh and Foster, Cornea 8:45, 1989) and is clearly in an undesirable state. In this respect, no lectin cell mitogenicity was observed in epithelial cells, so the clinical potential is more attractive for galectin-3 and galectin-7 than for growth factors. Over the last decade or so, the possibility of excimer laser keratotomy to correct corneal properties for the correction of myopia has become a reality. Thousands of such procedures are performed every week, and myopic people are freed from glasses and contact lenses. Focusing on the fact that 25% -30% of the world's adult population is short-sighted, it may be assumed that about 500,000 procedures per year are performed in the United States alone. After excimer laser surgery, epithelial healing may be delayed. Such delays should be avoided as much as possible, otherwise there is a risk that the cornea will suffer post-operative turbidity, infectious keratitis, and corneal tumors. Again, galectin based therapy promotes wound epithelialization in such cases.

<Other embodiments>
Other embodiments of the invention will be apparent to those of ordinary skill in the art in view of the practice or specification of the invention disclosed herein. The standards and examples are merely illustrative and the precise scope of the invention is indicated in the following claims.

Amino acid sequence and human galectin-3 are shown. (Genbank accession number BAA22164, SEQ ID NO: 1) Amino acid sequence and human galectin-7 are shown. (Genbank accession number I55469, SEQ ID NO: 2) Human Galectin 3 (SEQ ID NO: 1), Rabbit Galectin-3 Amino Acid Sequence (Genbank Accession Number JC4300), Chicken Galectin-3 Amino Acid Sequence (Genbank Accession Number AAB02856), and Hamster Galectin-3 Amino Acid The arrangement (Genbank accession number CAA55479) is arranged vertically using clustered W. The first (upper) sequence in the figure is amino acids 1 to 250 of human galectin-3 (SEQ ID NO: 1), the second sequence in the figure is amino acids 1 to 245 of hamster galectin-3, the third in the figure Is the amino acid 1 to 242 of rabbit galectin-3, and the fourth (lower) sequence in the figure is amino acid 1 to 262 of chicken galectin-3. A human galectin-7 (SEQ ID NO: 2), a rat galectin-7 (Genbank accession number P97590) and a mouse galectin-7 (Genbank accession number O54974) are vertically arranged using clustered W. The first (upper) sequence in the figure is amino acids 1 to 136 of rat galectin-7, the second sequence in the figure is amino acids 1 to 136 of mouse galectin-7, and the third (lower) sequence in the figure is Amino acids 1 to 136 of human galectin-7 (SEQ ID NO: 2). It is the list of the result of having scanned human galectin-3 (sequence number 1) by PROSITE. It is the list of the result of having scanned human galectin-7 (sequence number 2) by PROSITE. The galactoside-binding region of human galectin-3 and the consensus amino acid sequence (PF00337) derived from the hidden Markov model (HMM) in PFAM are shown vertically. The upper sequence is a common amino acid sequence (PF00337, SEQ ID NO: 3), and the lower amino acid sequence corresponds to amino acids 117 to 247 of SEQ ID NO: 1. A galactoside-binding region of human galectin-7 and a consensus amino acid sequence (PF00337) derived from a hidden Markov model (HMM) in PFAM are shown as vertically arranged. The upper sequence is a common amino acid sequence (PF00337, SEQ ID NO: 3), and the lower amino acid sequence corresponds to amino acids 5 to 135 of SEQ ID NO: 2. Includes a series of photographs of 2 mm exfoliation of the cornea to the extent that it can be healed in vivo, or an excimer laser wound on the cornea, and then analyzed for galectin-3 immunoreactivity in paraffin sections. (A) is a cornea colored with mematoxylin and eosin (i) normal cornea, (ii) cornea just after peeling, and (iii) cornea just after giving an excimer laser wound. (B) Immunohistologically colored (i) normal gal-3 ^{+ / +} cornea, (ii) healing gal-3 ^{+ / +} cornea after excimer laser wound. Colored immunohistologically (iii) normal gal-3 ^{− / −} cornea and (iv) healing gal-3 ^{− / −} cornea after excimer laser wounding. Dark color indicates positive immunostaining. WE, (wound edge), LE (leading edge of migrating epithelium), ie arrow (epithelium), arrowhead (white blood cells / stromal cells). 2 is a graph illustrating the effect of β-lactose (Lac) and saccharose (Suc) on the healing rate of wounded corneal epithelium. Wild-type (gal-3 ^{+ / +} ) and galectin-3-deficient (gal-3 ^-/- ) mice were given wounds that could be healed in vivo or in vitro using excimer laser or alkaline treatment The healing rate of the corneal epithelium is illustrated. The difference between galectin-7 gene expression and housekeeping gene selection between wild type (gal-3 ^{+ / +} ) and galectin-3 deficient (gal-3 ^{− / −} ) mice is illustrated. (GAPDH is D-glyceraldehyde 3-phosphate dehydrogenase, RPS29 is ribosomal protein S29, and ODC is ornithine decarboxylase.) The difference is determined by cDNA microarray and semi-quantitative PCR. The differences in expression of galectin-7 between wild type (gal-3 ^{+ / +} ) mice and galectin-3 deficient (gal-3 ^{− / −} ) mice are illustrated. The difference is determined by using (A) Western blot analysis, (B) immunohistochemical analysis, (C) mouse embryonic forebrain part. Exogenous effects on healing rate of wounded corneal epithelium of galectin-3-deficient (gal-3 ^{− / −} ) mice (FIG. 14A) and wild type (gal-3 ^{+ / +} ) mice (FIG. 14B) It is a graph which illustrates the influence of galectin-3. Illustrates the effect of β-lactose (Lac) and saccharose (Suc) on the healing rate of wounded corneal epithelium in wild type (gal-3 ^{+ / +} ) mice in the presence of exogenous galectin-3 It is a graph. (A) received wounds in wild type (gal-3 ^{+ / +} ) mice when exogenous galectin-7 was used alone or with β-lactose (Lac) or with saccharose (Suc) 2 is a graph illustrating the effect of exogenous galectin-7 on the healing rate of corneal epithelium. (B) shows the exogenous galectin-7 effect on the healing rate of wounded corneal epithelium in wild type (gal-3 ^{+ / +} ) mice and galectin-3 deficient (gal-3 ^{− / −} ) mice. It is a graph which compares influence. Figure 2 illustrates the amino acid sequence of human galectin-1 (SEQ ID NO: 6). Figure 2 illustrates the short amino acid sequence of human galectin-8 (SEQ ID NO: 4). The amino acid sequences of human galectin-1 (SEQ ID NO: 6), rat galectin-1, mouse galectin-1 and hamster galectin-1 are shown. Moreover, since they share a very high degree of identity, galectin-1 sequences are strongly conserved among mammalian species. The black part represents the position of the residue, which is not identical in all mammalian sequences. Rats and humans are 90.4% identical, mice and humans are 88.2% identical, and hamsters and humans are 91.2% identical. The figure also shows that the majority of residues at non-identical positions undergo conservative changes. For example, arginine is replaced with lysine at position 19, leucine is replaced with valine at position 24, serine is replaced with threonine at position 73, and aspartic acid is replaced with glutamic acid at position 136. The respective amino acid sequences of the short form (316 amino acids) of human galectin-8 (SEQ ID NO: 4) and the short form of mouse galectin-8, rat galectin-8, chicken galectin-8 and frog galectin-8 The corresponding amino acid sequence is shown. The amino acid sequence is aligned with the highlighted portion and represents a non-human amino acid sequence comparable to that of human and rat galectin-8. The three mammalian galectin-8 amino acid sequences share a very high degree of identity and are 78.2% identical, conserving amino acid changes in the majority of nonidentical parts (21.8%). ing. The amino acid changes are: phenylalanine at human position 20 comparable to tyrosine at similar positions in mouse and rat, glycine and serine at human position 22, glycine and rodent sequences at human position 26 It is an aspartate. Furthermore, since vertebrate species share significant identity of 43.0%, galectin-8 sequences have high identity among mammalian species and are conserved between vertebrate species. It is a bar graph which shows the influence which administered galectin to the dry eye syndrome in a mouse model. The syndrome was caused by injecting interleukin-1α (designated “IL-1” in the figure) on day 0 as a function of time. Galectin-3 was administered to each of five subjects per group using eye drops four times a day on days 1 and 2. The eye drops are at a concentration of 75 μg / ml or 150 μg / ml. Control group subjects were not injected with IL-1 and did not receive eye drops. Subjects in the “Buffer” group received the therapeutic agent in carrier medium only four times a day. Zoukhri, D.M. et. al. , Invest Ophthalmol Vis Sci 42 (5): 925-932, tear dynamics are assessed by fluorescein clearance. A lower value indicates better tear flow. When comparing the control group data with the treated group data with statistically significant difference of p <0.05 on the second day, the galectin treatment compared with the control animals not receiving galectin. The data show that tear dynamics have been restored to normal group tear dynamics.

Claims (8)

For treating dry eye, a mixed pharmaceutical composition for ocular administration, the compositions may contain pharmaceutically acceptable carriers or diluents, as well as provided with a galectin-3 protein, the galectin-3 protein promotes the diffusion of the tear film, in order to restore the kinetics of normal tears, the secretory mucins to be attached to the transmembrane mucin or glycoprotein on the conjunctiva and / or cornea valid Ryodea is,
The galectin-3 protein comprises a galectin-3 region rich in proline, glycine and tyrosine, and a galectin-3 galactoside binding region, and the galectin-3 region rich in proline, glycine and tyrosine is represented by SEQ ID NO: 1. Having an amino acid sequence of 60 to 140 amino acids with at least 60%, 70%, 80%, 90%, 95%, 99% or 100% identity to 15 to 116 amino acids, said galectin-3 The galactoside-binding region comprises an amino acid sequence of 80 to 180 amino acids, and the amino acid sequence has a bit score of at least 150 in a sequence comparison with the PFAM common sequence PF00337 (SEQ ID NO: 3) Composition. The xerophthalmia is xerostomia with recurrent epithelial erosion, the composition is mixed as an eye drop for topical administration as eye drops, and the composition is pH neutral, buffered The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is isotonic and comprises a thickener.   The pharmaceutical composition of claim 2, wherein the dry eye causes a wound, the wound being a corneal wound. The dry eye is, pharmaceutical composition according to claim 2, characterized in that caused by excimer laser keratectomy.   The pharmaceutical composition of claim 1, wherein the galectin-3 protein comprises the amino acid sequence of SEQ ID NO: 1. A container,
Pharmaceutically acceptable carrier or galectin the diluent - including 3 protein, for the treatment of dry eye, and a mixed composition for ocular administration, and instructions for use,
A kit for the treatment of dry eye comprising the galectin-3 protein promotes the diffusion of the tear film, in order to restore the kinetics of normal tears, the secretory mucins conjunctiva and / or take effect for Ryodea to bind to the transmembrane mucin or glycoprotein on the cornea,
The galectin-3 protein comprises a galectin-3 region rich in proline, glycine and tyrosine, and a galectin-3 galactoside binding region, and the galectin-3 region rich in proline, glycine and tyrosine is represented by SEQ ID NO: 1. Having an amino acid sequence of 60 to 140 amino acids with at least 60%, 70%, 80%, 90%, 95%, 99% or 100% identity to 15 to 116 amino acids, said galectin-3 The galactoside binding region includes an amino acid sequence of 80 to 180 amino acids, and the amino acid sequence has a bit score of at least 150 when compared with the PFAM common sequence PF00337 (SEQ ID NO: 3). . The kit according to claim 6 , wherein the protein is a single dose. The kit of claim 6 , wherein the composition is pH neutral, buffered, isotonic, and comprises a thickener.

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